The  upper 


UNITS. 


THB   METER   FO 
LENGTH.     .      . 


THE     GRAM 
\VEIGHT.      .      . 

THE     LITER    FO 
CAPACITY. 

Divisions  of 
o.ooi  ;  Micro,  on 

Multiples  at 
times  ;  myrta,  ic 


eter  :  Micron  (/ 

(g  166). 

long  distances. 


cries,  etc. 
than  the  correct 

centi,  o.oi  ;  Milli, 
too  times  ;  kilo,  1000 

ES 


>, ooo  microns  (ju) 


Meter  (Unit* 

39.3704  it          ,  m 

Centimeter  (cm.  )=io  millimeters  ;  10,000  microns  (//)  o.oi  meter  ;  0.3937  (I)  inch. 
Millimeter  (mm.  )=i,  ooo  microns  (//)  ;  o.i  cm.  ;  o.ooi  meter  ;  0.03937  (-fa)  inch. 
Micron  (u)  (Unit  of  measure  in   micrometry  (g  i66)=o.ooi  millimeter;  one  mil- 

lionth of  a  meter  ;  0.00003937  (  ^^)  inch. 
Yard=3  feet  ;  36  inches  ;  0.91439  meter  ;  91.4399  centimeters. 
Foot=i2  inches  ;  30.4799  centimeters  ;  304.799  millimeters. 
Inch—  y1^  foot  ;  3^  yard  ;  25.3999  millimeters  (2.54  centimeters). 
Liter  (Unit  of  capacity  )=  1,000  cubic  centimeters  (milliliters)  ;  (i  quart—.) 
Cubic  centimeter=o.ooi  liter  (milliliter)  ;  (TL  cub.  inch.) 
Fluid  ounce  (8  fluidrachms)=29.574  cubic  centimeters  (30  cc.  ). 
Gram  (Unit  of  weight  )=  i  cc.  of  water;  15.432  grains. 
Kilogram=n,  ooo  grams   ;  2.2046  (2^)  Ibs.  avoirdupois. 
Ounce  avoirdupois=437^  grains  ;  28.349  grams.  1 

Ounce  Troy  or  apothecaries—  480  grains  ;  31.103  grams  J    ^°  &ra:ns>  approx. 

TEMPERATURE 

To  change  Centigrade  to  Farenheit  :  (C.X  D+32  =  F. 
the  equivalent  of  10°  Centigrade,  C.=  io°X  f-f  32  =  50°  F. 

To  change  Farenheit  to  Centigrade  :  (F.—  32°)  X  f  —  C.     For  example  to  re- 
duce 50°  Farenheit  to  Centigrade,  F.=  5o°,  and  (50°—  32°)X  f  =  ioC.  ;   or  —  40 

' 


For  example,  to  find 


Farenheit 


. 
to    Centigrade,     F.=  —  40°    (—40°—  32°)=  —  72°,'   whence  — 


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S.  AGNES  SCHOOL 
LIBRARY. 


PRESENTED  ,8Y 


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zroscopes 

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THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 


PRESENTED  BY 

PROF.  CHARLES  A.  KOFOID  AND 
MRS.  PRUDENCE  W.  KOFOID 


THE  MICROSCOPE 

AN 

INTRODUCTION  TO   MICROSCOPIC 
METHODS    AND    TO    HISTOLOGY 

BY    SIMON    HENRY    GAGE 


PROFESSOR  OF  MICROSCOPY,  HISTOL- 
OGY AND  EMBRYOLOGY  IN  CORNELL 
UNIVERSITY,  AND  THE  NEW  YORK 
STATE  VETERINARY  COLLEGE. 


NINTH 


EDITION 


REVISED,    ENLARGED    AND    ILLUSTRATED   BY   OVER 
TWO   HUNDRED    FIGURES 

COMSTOCK  PUBLISHING  COMPANY 

ITHACA,   NEW   YORK 
1904 


Copyright,  1903. 

BY  SIMON  HENRY  GAGE. 

All  Rights  Reserved. 


Press  of 

THE   ITHACA  JOURNAL 
Ithaca,  N.  Y. 


PREFACE  TO  THE  EIGHTH  EDITION 


AS  the  preface  to  the  sixth  edition  of  this  work  expresses  accurately  what 
should  be  said  to-day  it  is  appended  : 

"The  rapid  advance  in  microscopical  knowledge,  and  the  great  strides  in  the 
sciences  employing  the  microscope  as  an  indispensable  tool,  have  reacted  upon  the 
microscope  itself,  and  never  before  were  microscopes  so  excellent,  convenient  and 
cheap.  Indeed,  the  financial  reason  for  not  possessing  a  microscope  can  no  longer 
be  urged  by  any  high  school  or  academy,  or  by  any  person  whose  profession 
demands  it. 

Naturally,  to  get  the  greatest  good  from  instruments,  tools,  or  machines  of  any 
kind,  the  one  who  uses  them  must  understand  the  principles  upon  which  their 
action  depends,  their  possibilities  and  limitations. 

That  the  student  may  acquire  a  just  comprehension  of  some  of  the  funda- 
mental principles  of  the  microscope,  and  gain  a  working  acquaintance  with  it  and 
its  applications,  this  book  has  been  prepared.  It  is  a  growth  of  the  laboratory,  and 
has  been  modified  from  time  to  time  to  keep  pace  with  optical  improvements  and 
advancing  knowledge. ' ' 

In  rewriting  this  edition  the  different  chapters  have  been  recast,  new  figures 
added  and  in  most  cases  the  matter  considerably  increased.  A  new  chapter  has 
been  added  upon  class  demonstrations.  The  general  availability  of  the  constant 
electric  current,  and  the  improvement  in  apparatus  have  made  micro-projection 
practicable  and  satisfactory.  It  has  served  the  writer  so  well  in  his  teaching  of 
histology  and  embryology  that  it  seemed  worth  while  to  give  the  benefit  of  his 
experience  to  his  fellow  workers. 

It  is  hoped  that  the  book  as  it  now  stands  will  serve  more  completely  than 
ever  before  the  needs  of  the  class-room  and  of  the  laboratory. 

"Simply  reading  a  work  on  the  microscope,  and  looking  a  few  times  into  an 
instrument  completely  adjusted  by  another,  is  of  very  little  value  in  giving  real 
knowledge.  In  order  that  the  knowledge  shall  be  made  alive,  it  must  become  a 
part  of  the  student's  experience  by  actual  experiments  carried  out  by  the  student 
himself.  Consequently,  exercises  illustrating  the  principles  of  the  microscope 
and  the  methods  of  its  employment  have  been  made  an  integral  part  of  the  work. 

"In  considering  the  real  greatness  of  the  microscope,  and  the  truly  splendid 
service  it  has  rendered,  the  fact  has  not  been  lost  sight  of  that  the  microscope  is, 
after  all,  only  an  aid  to  the  eye  of  the  observer,  only  a  means  of  getting  a  larger 
image  on  the  retina  than  would  be  possible  without  it ;  but  the  appreciation  of 


ivi3484±0 


IV 


PREFACE 


the  retinal  image,  whether  it  is  made  with  or  without  the  aid  of  a  microscope, 
must  always  depend  upon  the  character  and  training  of  the  seeing  and  appreciat- 
ing brain  behind  the  eye.  The  microscope  simply  aids  the  eye  in  furnishing  raw 
material,  so  to  speak,  for  the  brain  to  work  upon.  (From  3d  ed.) 

Grateful  acknowledgment  is  made  to  the  opticians  and  instrument  makers 
for  the  loan  of  cuts  and  for  courteous  and  complete  answers  to  numerous  questions  ; 
to  the  directors  of  laboratories  in  different  parts  of  the  country,  to  his  colleagues 
in  the  departments  of  Physics,  Chemistry  and  Electrical  Engineering  in  Cornell 
University  ;  and  finally  to  his  pupils  past  and  present  who  have  given  their  support 
and  encouragement. 

Inclosing  I  would  like  to  urge  those  who  are  interested  in  Microscopy  to  take 
some  microscopical  journal,  and  if  possible  to  become  a  member  of  some  micro- 
scopical club  or  society.  One  can  do  very  little  alone,  but  by  helping  others 
and  being  helped  in  return,  the  workers  in  any  field  of  human  endeavor  can 
accomplish  great  things. 

SIMON  HENRY  GAGE, 
CORNEI,!,  UNIVERSITY, 

October  /,  1901.  ITHACA,  N.  Y. ,  U.  S.  A. 


PREFACE  TO  THE  NINTH  EDITION 


In  this  edition  important  changes  have  been  made  on  pp.  7-8,  to  the  parts 
relating  to  serial  sections  and  to  micro-chemistry. 

The  chapter  on  the  projection  microscope  has  been  entirely  rewritten  and 
much  more  fully  illustrated.  With  the  good  apparatus  now  available  it  is  hoped 
that  teachers  will  make  more  use  of  this  excellent  method  of  demonstration. 

Due  to  the  courtesy  of  the  manufacturers  excellent  pictures  of  the  latest 
microscopes  and  apparatus  are  presented  in  this  edition.  It  can  but  be  a  cause  for 
rejoicing  to  the  multitudes  who  now  use  the  microscope  to  see  the  beauty  of  form 
which  it  has  attained. 

February  /, 


CONTENTS 


CHAPTER  I 

PAGE 

\     i-  59 — The  Microscope  and  its  parts i-  33 

CHAPTER  II 

$  60-128 — Lighting  and  Focusing  ;  Manipulation  of  Dry,  Adjustable 
and  Immersion  objectives  ;  Care  of  the  Microscope  and  of 
the  Eyes.  Laboratory  Microscopes 34-  89 

CHAPTER  III 
$  129-153 — Interpretation  of  Appearances 90-102 

CHAPTER  IV 
\  154-176 — Magnification  and  Micrometry 103-121 

CHAPTER  V 
\  177-187 — Drawing  with  the  Microscope 122-133 

CHAPTER  VI 

|   188-233 — The  Microspectroscope  and  Polariscope  ;  Micro-Chemistry  ; 

Textile  Fibers  and  Food  Products  ;  Micro-Metallography 134-160 

CHAPTER  VII 

I  234-335 — Slides  and  Cover-Glasses  ;  Mounting  ;  Isolation  ;  Sectioning 
by  the  Collodion  and  the  Paraffin  Methods  ;  Serial  Sections  ; 
Labeling  and  Storing  Microscopical  Preparations  ;  Reagents 
and  their  Preparation 161-204 

CHAPTER  VIII 

\  336-392 — Photographing  objects  with  a  Vertical  Camera  ;  Photograph- 
ing Large  Transparent  Objects  ;  Photographing  with  a  Mi- 
croscope ;  (A)  Transparent  Objects  ;  (B)  Opaque  Objects, 
and  the  Surfaces  of  Metals  and  Alloys  ;  Enlarging  Nega- 
tives ;  Photographing  Petri  Dishes  and  Culture  Tubes 205-242 

CHAPTER  IX 
$  393-426 — Class-Room  Demonstrations  with  the  Microscope  ;  With  the 

Projection  Microscope;  with  the  Episcope 243-267 

CHAPTER  X 

I  427-438 — The  Abbe  Test-Plate  and  Apertometer  ;  Equivalent  Focus  of 
Objectives  and  Oculars  ;  Drawings  for  Photo-Engravings  ; 
Wax  Models  ;  Some  Apparatus  for  Imbedding  and  Sec- 
tioning   268-281 

BOOKS  AND  PERIODICALS 282-288 

INDEX 289 


THE  MICROSCOPE  IN  SECTION 


1.  Huygenian  ocular  (see  p.  102  for  positive 

ocular). 

2.  Draw-tube  by  which  the  tube  is  length- 

ened or  shortened 

3.  Main  tube  or  body  containing  the  draw- 

tube,  and  attached  to  the  pillar  by  the 
arm. 

4.  Society  screw  in  the  lower  end  of  the 

draw-tube. 

5.  Society  screw  in  the  lower  end  of  the 

tube. 
5.     Objective  in  position. 

7,  Stage,  under  which  is  the  substage  with 

the  substage  condenser. 

8.  Spring  clip  for  holding  the  specimen. 


g.     Screw  for  centering,  and  handle  for  the 
iris  diaphragm  in  the  achromatic  con- 
denser (see  Fig.  41). 

10.  Iris    diaphragm    outside    the    principal 

focus  of  the  condenser  for  use  in  cen- 
tering (§  81), 

11.  Mirror  with  plane  and  concave  faces. 

12.  Horse-shoe  base. 

13     Rack  and  pinion  for  the  substage  con- 
denser. 

14.  Jointed  pillar. 

15.  Part  of  pillar  with  spiral  spring  of  fine 

adjustment. 

16.  Screw  of  fine  adjustment. 

17.  Milled  head  of  coarse  adjustment. 


THE  MICROSCOPE 


AND 


MICROSCOPICAL  METHODS 


CHAPTER   I 


THE   MICROSCOPE   AND   ITS    PARTS 


APPARATUS   AND   MATERIAL   FOR   THIS    CHAPTER 

A  simple  microscope  ($  2,  u) ;  A  compound  microscope  with  nose-piece  (Figs. 
70-80);  eye-shade  (Fig.  60),  achromatic  (g  20),  apochromatic  (^22),  dry  (§17), 
immersion  ($  18),  unadjustable  and  adjustable  objectives  ($  23,  24)  ;  Huygenian  or 
negative  ($38),  positive  (§37)  and  compensation  oculars  ($39)  ;  stage  microme- 
ter (Ch.  IV)  ;  homogeneous  immersion  liquid  (§  18)  ;  mounted  letters  or  figures 
($  53)  ;  ground-glass  and  lens  paper  (g  53). 

A    MICROSCOPE 

I  i.  A  Microscope  is  an  optical  apparatus  with  which  one  may  obtain  a  clear 
image  of  a  near  object,  the  image  being  always  larger  than  the  object  ;  that  is,  it 
enables  the  eye  to  see  an  object  under  a  greatly  increased  visual  angle,  as  if  the 
object  were  brought  very  close  to  the  eye  without  affecting  the  distinctness  of 
vision.  Whenever  the  microscope  is  use,d  for  observation,  the  eye  of  the  observer 
forms  an  integral  part  of  the  optical  combination  (Figs.  16,  21). 

$  2.  A  Simple  Microscope. — With  this  an  enlarged,  erect  image  of  an  object 
may  be  seen.  It  always  consists  of  one  or  more  converging  lenses  or  lens-systems 
(Figs.  16-20),  and  the  object  must  be  placed  within  the  principal  focus  (§  n). 
The  simple  microscope  may  be  held  in  the  hand  or  it  may  be  mounted  in  some 
way  to  facilitate  its  use  (Figs  17-20). 


MICROSCOPE  AND  ACCESSORIES 


[CH.  I 


FIGS.  1-9,  showing  the  Principal  Optic  Axis  and  the  Optical  Center  of  various 
forms  of  Lenses. 

Axis.  The  Principal  Optic  Axis.  c-cf .  Centers  of  curvature  of  the  two  sur- 
faces of  the  lens,  c.l.  Optical  center  of  the  lens,  r-r' '.  Radii  of  curvature  of 
the  two  lens  surfaces,  t-t' '.  Tangents  in  Fig.  4. 


$  3.  Principal  Optic  Axis. — In  spherical  lenses,  i.  e.,  lenses  whose  surfaces 
are  spherical,  the  Axis  is  a  line  joining  the  centers  of  curvature  and  indefinitely 
extended.  In  the  lens  it  is  the  unbroken  part  of  the  line  c-cf  in  the  figures.  In 
lenses  with  one  plane  surface  (Figs.  3,  6,  7)  the  radius  of  the  plane  surface  is  any 
line  at  right  angles  to  it;  but  in  determining  the  axis  it  must  be  the  one  which  is 
continuous  with  the  radius  of  the  curved  surface,  consequently  the  axis  in  such 
lenses  is  on  the  radius  of  the  curved  surface  which  meets  the  plane  surface  at  right 
angles. 

\  4.  Optical  Center. — The  optical  center  of  a  lens  is  the  point  through  which 
rays  pass  without  angular  deviation,  that  is,  the  emergent  ray  is  parallel  to  the 


\_CH.  I 


MICROSCOPE  AND  ACCESSORIES 


incident  ray.  It  is  determined  geometrically  by  drawing  parallel  radii  of  the 
curved  surfaces,  r-r'  in  Figs.  4-9,  and  joining  the  peripheral  ends  of  the  radii. 
The  optical  center  is  the  point  on  the  axis  cut  by  the  line  joining  the  peripheral 
ends  of  the  parallel  radii  of  the  two  lens  surfaces.  In  Figs.  4-5  it  is  within  the 
lens  ;  in  6-7  it  is  at  the  curved  surface,  and  in  the  meniscus  (8,  9)  it  is  wholly  out- 
side the  lens,  being  situated  on  the  side  of  the  greater  curvature. 

In  determining  the  center  in  a  lens  with  a  plane  surface,  the  conditions  can 
be  satisfied  only  by  using  the  radius  of  the  curved  surface  which  is  continuous 
with  the  axis  of  the  lens,  then  any  line  at  right  angles  to  the  plane  surface  will 
be  parallel  with  it,  and  may  be  considered  part  of  the  radius  of  the  plane  surface. 
(That  is,  a  plane  surface  may  be  considered  part  of  a  sphere  with  infinite  radius, 
hence  any  line  meeting  the  plane  surface  at  right  angles  may  be  considered  as  the 
peripheral  part  of  the  radius. )  In  Figs.  6,  7,  (r'}  is  the  radius  of  the  curved  sur- 
face and  (r)  of  the  plane  surface  ;  and  the  point  where  a  line  joining  the  ends  of 
these  radii  crosses  the  axis  is  at  the  curved  surface  in  each  case. 

By  a  study  of  Fig.  4  it  will  be  seen  that  if  tangents  be  drawn  at  the  peripheral 
ends  of  the  parallel  radii,  the  tangents  will  also  be  parallel  and  a  ray  incident  at 
one  tangential  point  and  traversing  the  lens  and  emerging  at  the  other  tangential 
point  acts  as  if  traversing,  and  is  practically  traversing  a  piece  of  glass  which  has 
parallel  sides  at  the  point  of  incidence  and  emergence,  therefore  the  emergent  ray 
will  be  parallel  with  the  incident  ray.  This  is  true  of  all  rays  traversing  the  center 
of  the  lens. 

Thick  Lenses. — In  all  of  the  diagrams  of  lenses  and  the  course  of  rays  through 
them  in  this  book  the  lenses  are  treated  as  if  they  were  infinitely  thin.  In  thick 
lenses  like  those  figured,  while  there  would  be  no  angular  deviation  for  rays  trav- 
ersing the  center  of  the  lens,  there  would  be  lateral  displacement.  This  is  shown 
in  Fig.  57  illustrating  the  effect  of  the  cover-glass. 

\  5.  Secondary  Axis. — Every  ray  traversing  the  center  of  the  lens,  except  the 
principal  axis,  is  a  secondary  axis  ;  and  every  secondary  axis  is  more  or  less 
oblique  to  the  principal  axis.  In  Fig.  14,  line  (2),  is  a  secondary  axis,  and  in  Fig. 
15,  line  (i).  See  also  Fig.  58. 


FIGS,  ro,  ii. — Sectional  views  of  a 
concave  or  diverging  and  a  convex  or 
converging  lens  to  show  that  in  the  con- 
cave lens  the  principal  focus  is  virtual  as 
indicated  by  the  dotted  lines,  while  with 
the  convex  lens  the  focus  is  real  and  on 
the  side  of  the  lens  opposite  to  that  from 
which  the  light  comes. 


\  6.  Principal  Focus. — This  is  the  point  where  rays  parallel  with  the  axis  and 
traversing  the  lens  cross  the  axis  ;  and  the  distance  from  the  focus  to  the  center  of 
the  lens  measured  along  the  axis  is  the  Principal  Focal  Distance.  In  the  diagrams, 
Fig.  10  is  seen  to  be  a  diverging  lens  and  the  rays  cross  the  axis  only  by  being  pro- 
jected backward.  Such  a  focus  is  said  to  be  virtual,  as  it  has  no  real  existence.  In 


MICROSCOPE  AND  ACCESSORIES 


\CH.  I 


Fig.  1 1  the  rays  do  cross  the  axis  and  the  focus  is  said  to  be  real.  If  the  light 
came  from  the  opposite  direction  it  would  be  seen  that  there  is  a  principal  focus 
on  the  other  side,  that  is  there  are  two  principal  foci,  one  on  each  side  of  the  lens. 
These  two  foci  are  both  principal  foci,  but  they  will  be  equally  distant  from  the 
center  of  the  lens  only  when  the  curvature  of  the  two  lens  surfaces  are  equal. 
There  may  be  foci  on  secondary  axes  also,  each  focus  on  a  secondary  axis  has  its 
conjugate.  In  the  formation  of  images  the  image  is  the  conjugate  of  the  object 
and  conversely  the  object  is  the  conjugate  of  the  image. 


W A 


FIG.  12. — Double  Convex  Lens,  Showing  Chromatic  Aberration. 

The  ray  of  white  light  (w)  is  represented  as  dividing  into  the  short  waved,  blue 
(b)  and  the  long  waved,  red  (r}  light.  The  blue  (b)  ray  comes  to  a  focus  nearer 
the  lens  and  the  red  ray  ( r}  farther  from  the  lens  than  the  principal  focus  ( J) . 
Principal  focus  (f)  for  rays  very  near  the  axis  ;  f  and  f" ',  foci  of blue  and  red 
light  coming  from  near  the  edge  of  the  lens.  The  intermediate  wave  lengths 
would  have  foci  all  the  way  between  ff  and  ff/ . 

\  7.  Chromatic  Aberration. — This  is  due  to  the  fact  that  ordinary  light  con- 
sists of  waves  of  varying  length,  and  as  the  effect  of  a  lens  is  to  change  the  direc- 
tion of  the  waves,  it  changes  the  direction  of  the  short  waves  more  markedly 
than  the  long  waves.  Therefore,  the  short  waved,  blue  light  will  cross  the  axis 
sooner  than  the  long  waved,  red  light,  and  there  will  result  a  superposition  of 
colored  images,  none  of  which  are  perfectly  distinct  (Fig.  12). 

FIG.  13.  The  ray  (o)  near  the 
edge  of  the  lens  is  brought  to  a 
focus  nearer  the  lens  than  the 
ray  (i).  Both  are  brought  to 
a  focus  sooner  than  rays  very 
near  the  axis,  (f)  Principal 
focus  for  rays  very  near  the 
axis;  (f}  Focus  for  the  ray 
(i),  and  (f")  Focus  for  the  ray 

(o).     Intermediate  rays  would 
FIG.  13.     Double  Convex  Lens,  showing  .,  ,,  ,,    '        r 

6          cross  the  axis  all  the  way  from 
Spherical  Aberration.  ( ff  t    f\ 

%  8.  Spherical  Aberration.— This  is  due  to  the  unequal  turning  of  the  light 
in  different  zones  of  a  lens.  The  edge  of  the  lens  refracts  proportionally  too 
much  and  hence  the  light  will  cross  the  axis  or  come  to  a  focus  nearer  the  lens 
than  a  ray  which  is  nearer  the  middle  of  the  lens.  Thus,  in  Fig.  13,  if  the  focus 


CH.I-] 


MICROSCOPE  AND  ACCESSORIES 


of  parallel  rays  very  near  the  axis  is  at/,  rays  (o  i),  nearer  the  edge,  would  come  to 
a  focus  nearer  the  lens,  the  focus  of  the  ray  nearest  the  edge  being  nearest  the  lens. 

\  9.  Correction  of  Chromatic  and  of  Spherical  Aberration. — Every  simple 
lens  has  the  defect  of  both  chromatic  and  spherical  aberration,  and  to  overcome 
this,  kinds  of  glass  of  different  refractive  power  and  different  dispersive  power 
are  combined,  concave  lenses  neutralizing  the  defects  of  convex  lenses.  If  the 
concave  lens  is  not  sufficiently  strong  to  neutralize  the  aberrations  of  the  convex 
lens,  the  combination  is  said  to  be  under-corrected,  while  if  it  is  too  strong  and 
brings  the  marginal  rays  or  the  blue  rays  to  a  focus  beyond  the  true  principal 
focus,  the  combination  is  over-corrected. 

In  Newton's  time  there  was  supposed  to  be  a  direct  proportion  between  the 
refractive  power  of  any  transparent  medium  and  its  dispersive  power  ( i.  e.  its  power 
to  separate  the  light  into  colors).  If  this  were  true  then  the  contention  of  Newton 
that  it  would  be  impossible  to  do  away  with  the  color  without  al  the  same  time 
doing  away  with  the  refraction  would  be  true  and  useful  achromatic  combinations 
would  be  impossible.  It  was  found  by  experiment,  however,  that  there  is  not  a 
direct  ratio  between  the  refractive  and  dispersive  powers  for  the  different  colors 
in  different  forms  of  glass,  so  that  it  is  possible  to  do  away  largely  with  chromatic 
aberration  and  retain  sufficient  refraction  to  make  the  combination  serve  for  the 
production  of  images.  ( See  also  the  discussion  under  apochromatic  objectives  \  22  ) 

Probably  no  higher  technical  skill  is  used  in  any  art  than  is  requisite  in  the 
preparation  of  microscopical  objectives,  oculars  and  illuminators. 

FIGS.  14  AND  15.  14.  Convex  lens 
showing  the  position  of  the  object  ( A-B ) 
outside  the  principal  focus  (F],  and 
the  course  of  the  rays  in  the  formation 
of  real  images.  To  avoid  confusion  the 
rays  are  drawn  from  only  one  point. 

A  B.  Object  outside  the  principal 
focus.  Bf  Af .  Real,  enlarged  image 
on  the  opposite  side  of  the  lens. 

Axis.  Principal  optic  axis.  1,2,3. 
Rays  after  traversing  the  lens.  They 
are  converging,  and  consequently  form 
a  real  image.  The  dotted  line  and  the 
line  (2}  give  the  direction  of  the  rays  as 
if  unaffected  by  the  lens.  (/^).  The 
principal  focus. 

FIG.  15. — Convex  lens,  showing  the 
position  of  the  object  (A  B}  within  the 
principal  focus  and  the  course  of  rays 
in  the  formation  of  a  virtual  image. 

A  B.  The  object  placed  between  the  lens  and  its  focus  ;  A'  B'  virtual  image 
formed  by  tracing  the  rays  backward.  It  appears  on  the  same  side  of  the  lens  as 
the  object,  and  is  erect  (\  //). 

Axis.     The  principal  optic  axis  of  the  lens.     F.   The  principal  focus. 

i,  2,  j.  Rays  from  the  point  B  of  the  object.  They  are  diverging  after  trav- 
ersing the  lens,  but  not  so  divergent  as  if  no  lens  were  present,  as  is  shown  by  the 


MICROSCOPE  AND  ACCESSORIES 


[CH.  I 


dotted  lines.     Ray  (/)  traverses  the  center  of  the  lens,  and  is  therefore  not  deflected. 
It  is  a  secondary  axis  ($5). 

\  10.  Geometrical  Construction  of  Images. — As  shown  in  Figs.  14-15,  for  the 
determination  of  any  point  of  an  image,  or  the  image  being  known,  to  determine 
the  corresponding  part  of  the  object,  it  is  necessary  to  know  the  position  of  the 
principal  focus  (and  there  is  one  on  each  side  of  the  lens,  $6),  and  the  optical 
center  (Figs.  1-9  of  the  lens).  Then  a  secondary  axis  (2)  in  Fig.  14,  (i)  in  Fig. 
15,  is  drawn  from  the  extremity  of  the  object  and  prolonged  indefinitely  above  the 
lens,  or  below  it  for  virtual  images.  A  second  line  is  drawn  from  the  extremity  of 
the  object,  (3)  in  Fig.  14,  (2)  in  Fig.  15,  to  the  lens  parallel  with  the  principal 
axis.  After  traversing  the  lens  it  must  be  drawn  through  the  principal  focal  point. 
If  now  it  is  prolonged  it  will  cross  the  secondary  axis  above  the  lens  for  a  real 
image  and  below  for  a  virtual  image.  The  crossing  point  of  these  lines  determines 
the  position  of  the  corresponding  part  of  the  image.  Commencing  with  any  point 
of  the  object  the  corresponding  point  of  the  image  may  be  determined  as  just 
described,  and  conversely  commencing  with  the  image,  corresponding  points  of 
the  object  may  be  determined. 

SIMPLE   MICROSCOPE  :    EXPERIMENTS 

§  ii.  Employ  a  tripod  or  other  simple  microscope,  and  for  object 
a  printed  page.  Hold  the  eye  about  two  centimeters  from  the  upper 
surface  of  the  magnifier,  then  alternately  raise  and  lower  the  magnifier 
until  a  clear  image  may  be  seen.  (This  mutual  arrangement  of  micro- 
scope and  object  so  that  a  clear  image  is  seen,  is  called  focusing). 
When  a  clear  image  is  seen,  note  that  the  letters  appear  as  with  the 
unaided  eye  except  that  they  are  larger,  and  the  letters  appear  erect  or 
right  side  up,  instead  of  being  inverted,  as  with  the  compound 
microscope  (§12). 

FIG.  1 6.  Diagram  of  the  simple  microscope  show- 
ing the  course  of  the  rays  and  all  the  images,  and 
that  the  eye  forms  an  integral  part  of  it. 

A*  B1.  The  object  within  the  principal  focus.  A* 
£3.  The  virtual  image  on  the  same  side  of  the  lens 
as  the  object.  It  is  indicated  with  dotted  lines,  as  it 
has  no  actual  existence. 

B2  A2.  Retinal  image  of  the  object  (A1  Bl).  The 
virtual  image  is  simply  a  projection  of  the  retinal 
image  in  the  field  of.  vision. 

Axis.  The  principal  optic  axis  of  the  micro- 
scope and  of  the  eye.  Cr.  Cornea  of  the  eye.  L. 
Crystalline  lens  of  the  eye.  R.  Ideal  refracting 
surface  at  which  all  the  refractions  of  the  eye  may 
be  assumed  to  take  place. 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


Hold  the  simple  microscope  directly  toward  the  sun  and  move  it 
away  from  and  toward  a  piece  of  printed  paper  until  the  smallest 
bright  point  is  obtained.  This  is  the  burning  point  or  focus  and  as 
the  rays  of  the  sun  are  nearly  parallel,  the  burning  point  represents 
approximately  the  principal  focus  (Fig.  n).  The  above  and  follow- 
ing operations  are  more  easily  accomplished  if  the  lens  is  supported  as 
in  Fig.  20. 

Without  changing  the  position  of  the  magnifier  or  paper  look  into 
the  magnifier,  holding  the  eye  close  to  the  upper  surface  and  the 
letters  on  the  paper  may  be  seen,  but  they  will  appear  much  sharper 
to  the  eyes  of  most  people  if  the  magnifier  is  brought  nearer  to  the 
paper,  that  is  so  that  the  printed  paper  is  within  the  principal  focal 
distance  (Fig.  15  and  16). 


Fig.  1 6  A.  Figures  of  a  normal  (emme- 
tropic],  a  far  sighted  (hyperopic)  and  a  short 
sighted  (myopic]  eye  to  show  that  when  the  eye 
is  at  rest  the  normal  eye  (E)  focuses  parallel 
rays  on  the  retina  while  the  far-sighted  eye  (//) 
focuses  parallel  rays  beyond  the  retina.  The 
short  sighted  eye  (M)  focuses  parallel  rays  in 
front  of  the  retina.  The  dotted  lines  show  that 
in  the  hyperopic  eye  the  rays  must  be  converging 
to  come  to  a  focus  on  the  retina  while  with  the 
myopic  eye  they  must  be  diverging. 


After  getting  as  clear  an  image  as  possible  by  focusing  the  simple 
microscope,  raise  the  magnifier  until  the  letters  are  at  a  distance  a 
little  greater  than  the  principal  focal  distance.  Look  into  the  magni- 
fier and  note  the  clearness  of  the  virtual  image,  then  slowly  elevate 
the  head  above  the  magnifier  and  when  the  eye  is  about  60  to  100 
centimeters  above  the  lens  a  real  image  can  be  seen.  That  is  an 
image  in  which  the  letters  are  inverted  as  with  the  objective  of  the 
compound  microscope  (see  §  53).  If  the  magnifier  is  raised  somewhat 
so  that  the  printed  letters  are  markedly  without  the  principal  focus  the 
real  image  will  be  seen  more  clearly  especially  if  the  eye  is  brought 
somewhat  near  the  magnifier.  The  above  experiments  show  two 
things. 


8 


MICROSCOPE  AND  ACCESSORIES 


\_CH.  I 


1 i )  That  every  convex  or  converging  lens  or  lens  system  can 
serve  to  form   either  a  virtual   or  a  real   image,    depending   upon    its 
position  with  reference  to  the  object. 

(2)  They  show  also  that  without  changing  the  position  of  the 
magnifier,  if   it  is  slightly  further   from   the  object  than  its  principal 
focal  distance,  either  a  virtual  image  or  a  real  image  may  be  seen  by 
many  people,    depending  upon    the  position  of  the  eye.      (a)  If  the 
eye  is  close  to  the  magnifier  an  enlarged  erect  virtual  image  will  be 
seen.       (b)  With    the   eye   at   a   considerable   distance   an    enlarged 
inverted  real  image  may  be  seen. 

Fig.  1 6  B.  Figure  to  show  that  with  a 
simple  microscope  if  the  object  is  slightly  be- 
yond Ihe  principal  focus -(F)  a  real  image 
will  be  formed  at  A'  which  can  be  seen  by  an 
eye  at  £,  and  that  if  a  normal  or  hyperopic 
eye  is  at  Ef  a  virtual  image  can  be  seen 
without  changing  the  position  of  the  simple 
microscope.  The  long-sighted  eye  can  see 
this  image  best  as  it  naturally  focuses  con- 
verging rays  on  the  retina.  The  myopic  eye 
either  sees  no  image  at  all,  or  a  mere  blur, 
depending  .upon  the  amount  of  myopia.  A. 
object;  A/  real  image  above  the  magnifier ; 
A."  virtual  image  which  can  be  seen  below 
the  lens  by  an  eye  at  E> ';  E.  eye  in  posi- 
tion to  see  a  real  image  ;  E.f  eye  in  position 
to  see  A"  a  virtual  image;  F.  principal 
focus  of  the  magnifier. 


FIG.   17.     Tripod  Magnifier. 

While  the  law  is  absolute  that  real  images  are  formed  only  when 
the  object  is  without  the  principal  focal  distance,  and  virtual  images 
only  when  the  object  is  within  the  focus,  the  above  experiments  show 


CH.I} 


MICROSCOPE  AND  ACCESSORIES 


8a 


FIG.  18.     Lens-holder   (The  Bausch  FIG.   19.       The  Hastings  Aplan- 

&  Lomb  Optical  Co.)  atic  Triplet.    (The  Bausch  &  Lomb 

Optical  Co. ) 


FIG.  20.     Dissecting  Microscope.     This  is  simply  a  device  for  holding  the  lens 
and  the  object  to  be  observed.     ( The  Bausch  &  Lomb  Optical  Co. ) 


8b 


MICROSCOPE  AND  ACCESSORIES 


[_CH.  I 


most  conclusively  that  the  eye  is  a  part  of  the  optical  arrangement 
when  the  microscope  is  actually  used  for  observation,  and  that  the 
microscope  with  the  eye  is  a  different  apparatus  from  the  microscope 
considered  by  itself. 

The  diagrams,  Figs.  16  A,  B,  are  introduced  to  show  under  what 
conditions  both  a  virtual  and  a  real  image  may  be  seen  without 
changing  the  position  of  the  magnifier  or  the  object. 


FIG.  17  A.  Diagrams  showing-  the  formation  of  real  and  of  virtual  images 
and  of  the  retinal  image  in  using  the  simple  microscope.  See  the  explanation  of 
Figs.  14,  75,  16. 

Simple  microscopes  are  very  convenient  when  only  a  small  mag- 
nification (Ch.  IV)  is  desired,  as  for  dissecting.  Achromatic  triplets 
are  excellent  and  convenient  for  the  pocket.  For  use  in  conjunction 
with  a  compound  microscope,  the  tripod  magnifier  (Fig.  17)  is  one  of 
the  best  forms.  For  many  purposes  a  special  mechanical  mounting  is 
to  be  preferred. 

COMPOUND  MICROSCOPE 

\  12.  A  Compound  Microscope. — This  enables  one  to  see  an  enlarged,  in- 
verted image.  It  always  consists  of  two  optical  parts — an  objective,  to  produce  an 
enlarged,  inverted,  real  image  of  the  object,  and  an  ocular  acting  in  general  like 
a  simple  microscope  to  magnify  this  real  image  (Fig.  21).  There  is  also  usually 
present  a  mirror,  or  both  a  mirror  and  some  form  of  condenser  or  illuminator  for 
lighting  the  object.  The  stand  of  the  microscope  consists  of  certain  mechanical 
arrangements  for  holding  the  optical  parts  and  for  the  more  satisfactory  use  of 
them.  (See  frontispiece. ) 

\  13.  The  Mechanical  Parts  of  a  laboratory,  compound  microscope  are  shown 
in  the  frontispiece,  and  are  described  in  the  explanation  of  that  figure.  The  stu- 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


dent  should  study  the  figure  with  a  microscope  before  him  and  become  thoroughly 
familiar  with  the  names  of  all  the  parts.  See  also  the  cuts  of  microscopes  at  the 
end  of  Ch.  II. 


OPTICAL   PARTS 

\  14.  Microscopic  Objective. — This 
consists  of  a  converging  lens  or  of  one 
or  more  converging  lens-systems,  which 
give  an  enlarged,  inverted,  real  image  of 
the  object  ( Figs.  14,  21 ).  And  as  for  the 
formation  of  real  images  in  all  cases, 
the  object  must  be  placed  outside  the 
principal  focus,  instead  of  within  it,  as 
for  the  simple  microscope.  (See  |§  n, 
53,  Figs.  1 6,  21.) 

Modern  microscopic  objectives  usu- 
ally consist  of  two  or  more  systems  or 
combinations  of  lenses,  the  one  next 
the  object  being  called  the  front  com- 
bination or  lens,  the  one  farthest  from 
the  object  and  nearest  the  ocular,  the 
back  combination  or  system.  There  may 
be  also  one  or  more  intermediate  sys- 
tems. Each  combination  is,  in  general, 
composed  of  a  convex  and  a  concave 
lens.  The  combined  action  of  the  sys- 
tem serves  to  produce  an  image  free 
from  color  and  from  spherical  distor- 
tion. In  the  ordinary  achromatic  ob-J 
jectives  the  convex  lenses  are  of  crown 
and  the  concave  lenses  of  flint  glass 
(Figs.  22,23).  . 

FIG.  21.  Diagram  showing  the 
principle  of  a  compound  microscope  with 
the  course  of  the  rays  from  the  object 
(A  B)  through  the  objective  to  the  real 
image  (B'  A'},  thence  through  the  ocu- 
lar and  into  the  eye  to  the  retinal  image 
(A*B2),  and  the  projection  of  the  retinal 
image  into  the  field  of  vision  as  the 
virtual  image  (B^A*). 

AB.  The  object.  A*B\  The  retinal 
image  of  the  inverted  real  image,  (BlAl) , 
formed  by  the  objective.  B^A*.  The 
inverted  virtual  image,  a  projection  of 
the  retinal  image. 


•c 


IO 


MICROSCOPE  AND  ACCESSORIES 


Axis.     The  principal  optic  axis  of  the  microscope  and  of  the  eye. 

Cr.  Cornea  of  the  eye.  L.  Crystalline  lens  of  the  eye .  R.  Single,  ideal,  re- 
fracting surface  at  which  all  the  refractions  of  the  eye  may  be  assumed  to  take 
place. 

F.  F.     The  principal  focus  of  the  positive  ocular  and  of  the  objective. 

Mirror.  The  mirror  reflecting  parallel  rays  to  the  object.  The  light  is  central. 
See  Ch.  II. 

Pos.  Ocular.  An  ocular  in  which  the  real  image  is  formed  outside  the  ocular. 
Compare  the  positive  ocular  with  the  simple  microscope  (Fig.  16). 

NOMENCLATURE   OR   TERMINOLOGY   OF   OBJECTIVES 

\  15.  Equivalent  Focus. — In  America,  England,  and  sometimes  also  on  the 
Continent,  objectives  are  designated  by  their  equivalent  focal  length.  This  length 
is  given  either  in  inches  (usually  contracted  to  in. )  or  in  millimeters  (mm.)  Thus: 
An  objective  designated  T^  in.  or  2  mmM  indicates  that  the  objective  produces  a 
real  image  of  the  same  size  as  is  produced  by  a  simple  converging  lens  whose 
principal  focal  distance  is  T^  inch  or  2  millimeters  (Fig.  u).  An  objective 
marked  3  in.  or  75  mm.,  produces  approximately  the  same  sized  real  image  as  a 
simple  converging  lens  of  3  inches  or  75  millimeters  focal  length.  And  in  accord- 
ance with  the  law  that  the  relative  size  of  object  and  image  vary  directly  as  their 
distance  from  the  center  of  the  lens  (Figs.  14,  15,  see  Ch.  IV,)  it  follows  that  the 
less  the  focal  distance  of  the  simple  lens  or  of  the  equivalent  focal  distance  of  the 
objective,  the  greater  is  the  size  of  the  real  image,  as  the  tube-length  remains  con- 
stant and  the  image  in  all  cases  is  found  at  about  i6oor  250  mm.  from  the  objective. 
§  16.  Numbering  or  Lettering  Objectives. — Instead  of  designating  objectives 
by  their  equivalent  focus,  many  Continental  opticians  use  letters  or  figures  for  this 
purpose.  With  this  method  the  smaller  the  number,  or  the  earlier  in  the  alpha- 
bet the  letter,  the  lower  is  the  power  of  the  objective.  (See  further  in  Ch.  IV,  for 
the  power  or  magnification  of  objectives).  This  method  is  entirely  arbitrary  and 
does  not,  like  the  one  above,  give  direct  information  concerning  the  objective. 

§  17.  Air  or  Dry  Objectives. — These  are  objectives  in  which  the  space  be- 
tween the  front  of  the  objective  and  the  object  or  cover-glass  is  filled  with  air 
(Fig.  22).  Most  objectives  of  low  and  medium  power  (i.  e,,  \  in.  or  3  mm.  and 
lower  powers)  are  dry. 

FIG.  22.  Section  of  a  dry  objective  showing 
working  distance  and  lighting  by  reflected 
light. 

Axis.  The  principal  optic  axis  of  the  ob- 
jective. 

B  C.     Back   Combination,  composed  of  a 
plano-concave  lens  of  flint  glass   (F},  and  a 
double  convex  lens  of  crown  glass  (c). 
F  C.     Front  Combination. 
C,  O,  si.     The  cover-glass,  object  and  slide. 
Mirror.    The  mirror  is  represented  as  above 
the  stage,  and  as  reflecting  parallel  rays  from 
its  plane  face  upon  the  object. 

Stage.  Section  of  the  stage  of  the  microscope. 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


n 


W.  The  Working  Distance,  that  is  the  distance  from  the  front  of  the  objective 
to  the  object  when  the  objective  is  in  focus. 

§  18.  Immersion  Objectives. — An  immersion  objective  is  one  with  which 
there  is  some  liquid  placed  between  the  front  of  the  objective  and  the  object  or 
cover-glass.  The  most  common  immersion  objectives  are  those  (A)  in  which 
water  is  used  as  the  immersion  fluid,  and  (B)  where  some  liquid  is  used  having  the 
same  refractive  and  dispersive  power  as  the  front  lens  of  the  objective.  Such  a 
liquid  is  called  homogeneous,  as  it  is  optically  homogeneous  with  the  front  glass  of 
the  objective.  It  may  consist  of  thickened  cedar  wood  oil  or  of  glycerin  contain- 
ing some  salt,  as  stannous  chlorid  in  solution.  When  oil  is  used  as  the  immersion 
fluid  the  objectives  are  frequently  called  oil  immersion  objectives.  The  disturb- 
ing effect  of  the  cover-glass  (Fig.  57)  is  almost  wholly  eliminated  by  the  use  of 
homogeneous  immersion  objectives,  as  the  rays  undergo  very  little  or  no  refraction 
on  passing  from  the  cover-glass  through  the  immersion  medium  and  into  the  ob- 
jective ;  and  when  the  object  is  mounted  in  balsam  there  is  practically  no  refrac- 
tion in  the  ray  from  the  time  it  leaves  the  balsam  till  it  enters  the  objective. 

FIG.  23.  Sectional  view  of  an  Immersion,  Ad- 
justable Objective,  and  the  object  lighted  with  axial 
or  central  and  with  oblique  light. 

Axis.     The  principal  optic  axis  of  the  objective. 

B  C,  M  C,  F  C.  The  back,  middle  and  front 
combination  of  the  objective.  In  this  case  the 
front  is  not  a  combination,  but  a  single  plano- 
convex lens. 

A,  B.  Parallel  rays  reflected  by  the  mirro* 
axially  or  centrally  upon  the  object. 

C.     Ray  reflected  to  the  object  obliquely. 

I.  Immersion  fluid  between  the  front  of  the 
objective  and  the  cover  glass  or  object  (O). 

Mirror.     The  mirror  of  the  microscope. 

O.  Object.  It  is  represented  without  a  cover- 
glass.  Ordinarily  objects  are  covered  whether  ex- 
amined with  immersion  or  with  dry  objectives. 

Stage.     Section  of  the  stage  of  the  microscope. 

\  19.  Non- Achromatic  Objectives. — These  are  objectives  in  which  the  chro- 
matic aberration  is  not  corrected,  and  the  image  produced  is  bordered  by  colored 
fringes.  They  show  also  spherical  aberration  and  are  used  only  on  very  cheap 
microscopes.  (\\  7,  8,  Figs.  12,  13). 

£  20.  Achromatic  Objectives. — In  these  the  chromatic  and  the  spherical  aber- 
ration are  both  largely  eliminated  by  combining  concave  and  convex  lenses  of  dif- 
ferent kinds  of  glass  "so  disposed  that  their  opposite  aberrations  shall  correct 
each  other."  All  the  better  forms  of  objectives  are  achromatic  and  also  aplanatic. 
That  is  the  various  spectral  colors  come  to  the  same  focus. 

|  21.  Aplanatic  Objectives,  etc.— These  are  objectives  or  other  pieces  of 
optical  apparatus  (oculars,  illuminators,  etc.),  in  which  the  spherical  distortion  is 


12  MICROSCOPE  AND  ACCESSORIES  [CM.  I 

wholly  or  nearly  eliminated,  and  the  curvatures  are  so  made  that  the  central  and 
marginal  parts  of  the  objective  focus  rays  at  the  same  point  or  level.  Such  pieces 
of  apparatus  are  usually  achromatic  also. 

§  22.  Apochromatic  Objectives. — A  term  used  by  Abbe  to  designate  a  form  of 
objective  made  by  combining  new  kinds  of  glass  with  a  natural  mineral  (Calcium 
fluorid,  Fluorite,  or  Fluor  spar).  The  name,  Apochromatic,  is  used  to  indicate 
the  higher  kind  of  achromatism  in  which  rays  of  three  spectral  colors  are  com- 
bined at  one  focus,  instead  of  rays  of  two  colors  as  in  the  ordinary  achromatic  ob- 
jectives. At  the  present  time  (1901)  several  opticians  make  apochrornatic  ob- 
jectives without  using  the  fluorite.  Some  of  the  early  apochromatics  deteriorated 
rather  quickly  in  hot  moist  climates.  Those  now  made  are  quite  permanent. 

The  special  characteristics  of  these  objectives,  when  used  with  the  "compen- 
sating oculars"  are  as  follows  : 

(1)  Three  rays  of  different  color  are  brought  to  one  focus,  leaving  a  small  ter- 
tiary spectrum  only,  while  with  objectives  as  formerly  made  from  crown  and  flint 
glass,  only  two  different  colors  could  be  brought  to  the  same  focus. 

(2)  In  these  objectives  the  correction  of  the  spherical  aberration  is  obtained 
for  two  different  colors  in   the  brightest  part  of  the  spectrum,  and  the  objective 
shows  the  same  degree  of  chromatic  correction  for  the  marginal  as  for  the  central 
part  of  the  aperture.     In  the  old  objectives,  correction  of  the  spherical  aberration 
was  confined  to  rays  of  one  color,  the  correction  being  made  for  the  central  part  of 
the  spectrum,  the  objective  remaining  under-corrected  spherically  for  the  red  rays 
and  <?zw-corrected  for  the  blue  rays  ($9). 

(3)  The  optical  and  chemical  foci  are  identical,  and  the  image  formed  by 
the  chemical  rays  is  much  more  perfect  than  with  the  old  objectives,  hence  the 
new  objectives  are  well  adapted  to  photography. 

(4)  These  objectives  admit  of  the  use  of  very  high  oculars,  and  seem  to  be  a 
considerable  improvement  over  those  made  in  the  old  way  with  crown  and  flint 
glass.     According  to   Dippel  (Z.  w.  M.  1886,  p.  300)  dry  apochrornatic  objectives 
give  as  clear  images  as  the  same  power  water  immersion  objectives  of  the  old  form. 

§  23.  Non-Adjustable  or  Unadjustable  Objectives. — Objectives  in  which  the 
lenses  or  lens  systems  are  permanently  fixed  in  their  mounting  so  that  their  rela- 
tive position  always  remains  the  same.  Low  power  objectives  and  those  with 
homogenous  immersion  are  mostly  non-adjustable.  For  beginners  and  those  un- 
skilled in  manipulating  adjustable  objectives  ($  24),  non- adjustable  ones  are  more 
satisfactory,  as  the  optician  has  put  the  lenses  in  such  a  position  that  the  most 
satisfactory  results  may  be  obtained  when  the  proper  thickness  of  cover-glass  and 
tube-length  are  employed.  (See  table  of  tube-length  and  thickness  of  cover-glass 
below,  p.  14.) 

§  24.  Adjustable  Objectives.— An  adjustable  objective  is  one  in  which  the  dis- 
tance between  the  systems  of  lenses  (usually  the  front  and  the  back  systems)  may 
be  changed  by  the  observer  at  pleasure.  The  object  of  this  adjustment  is  to  cor- 
rect or  compensate  for  the  displacement  of  the  rays  of  light  produced  by  the 
mounting  medium  and  the  cover-glass  after  the  rays  have  left  the  object.  It  is 
also  to  compensate  for  variations  in  "tube-length."  See  $29.  As  the  displace- 
ment of  the  rays  by  the  cover-glass  is  the  most  constant  and  important,  these  ob- 
jectives are  usually  designated  as  having  cover-glass  adjustment  or  correction. 
(Fig.  23.  See  also  practical  work  with  adjustable  objectives,  Ch.  II). 


CH.   7]  MICROSCOPE  AND  ACCESSORIES  13 

\  25.  Parachromatic,  Pantachromatic  and  Semi-apochromatic  Objectives. 
These  are  trade  names  for  objectives,  most  of  them  containing  one  or  more  lenses 
of  the  new  glass  (§22).  They  are  said  to  approximate  much  more  closely  to  the 
apochromatics  than  to  the  ordinary  objectives. 

\  26.  Variable  Objective. — This  is  a  low  power  objective  of  36  to  26  mm. 
equivalent  focus,  depending  upon  the  position  of  the  combinations.  By  means  of 
a  screw  collar  the  combinations  may  be  separated,  diminishing  the  power,  or 
approximated  and  thereby  increasing  it. 

$  27.  Projection  Objectives. — These  are  designed  especially  for  projecting 
an  image  on  a  screen  and  for  photo-micrography.  They  are  characterized  by  hav- 
ing a  flat,  sharp  field  brilliantly  lighted.  (See  Ch.  IV,  IX. ) 

\  28.  Illuminating  or  Vertical  Illuminating  Objectives. — These  are  designed 
for  the  study  of  opaque  objects  with  good  reflecting  surfaces,  like  the  rulings  on 
metal  bars  and  broken  or  polished  and  etched  surfaces  of  metals  employed  in 
micro- metallography.  The  light  enters  the  side  of  the  tube  or  objective  and  is 
reflected  vertically  downward  through  the  objective  and  thereby  is  concentrated 
upon  the  object.  The  object  reflects  part  of  the  light  back  into  the  microscope 
thus  enabling  one  to  see  a  clear  image. 

$  29.  Tube-Length  and  Thickness  of  Cover-Glasses. — "In  the  construction 
of  microscopic  objectives,  the  corrections  must  be  made  for  the  formation  of  the 
image  at  a  definite  distance,  or  in  other  words  the  tube  of  the  microscope  on 
which  the  objective  is  to  be  used  must  have  a  definite  length.  Consequently  the 
microscopist  must  know  and  use  this  distance  or  'microscopical  tube-length'  to 
obtain  the  best  results  in  using  any  objective  in  practical  work."  Unfortunately 
different  opticians  have  selected  different  tube-lengths  and  also  different  points 
between  which  the  distance  is  measured,  so  that  one  must  know  what  is  meant  by 
the  tube-length  of  each  optician  whose  objectives  are  used.  See  table. 

The  thickness  of  cover-glass  used  on  an  object  (See  Ch.  VII,  on  mounting), 
except  with  homogeneous  immersion  objectives,  has  a  marked  effect  on  the  light 
passing  from  the  object  (Fig.  57).  To  compensate  for  this  the  position  of  the  sys- 
tems composing  the  objective  are  closer  together  than  they  would  be  if  the  object 
were  uncovered.  Consequently,  in  non-adjustable  objectives  some  standard 
thickness  of  cover-glass  is  chosen  by  each  optician  and  the  position  of  the  systems 
arranged  accordingly.  With  such  an  objective  the  image  of  an  uncovered  object 
would  be  less  distinct  than  a  covered  one,  and  the  same  result  would  follow  the 
use  of  a  cover-glass  much  too  thick. 


•  *The  information  contained  in  the  tables  on  the  following  page  was  very 
kindly  furnished  by  the  opticians  named,  or  obtained  by  consulting  catalogs.  In 
most  of  the  later  catalogs  the  information  is  definite,  and  many  makers  now 
not  only  put  their  names  and  the  equivalent  focal  length  on  their  objectives, 
but  they  add  the  numerical  aperture  (§  31)  and  the  tube-length  for  which 
the  objective  is  corrected.  This  is  in  accordance  with  the  recommendations 
of  the  author  in  the  original  paper  on  "tube-length,"  (Proc.  Amer.  Soc. 
Micr.,  Vol.  IX.,  p.  168,  also  by  Bausch,  Vol.  XII,  p.  43).  If  the  table  in 
this  edition  is  compared  with  the  original  table  or  with  that  in  the  pre- 
vious editions  of  this  book  some  differences  will  be  noted,  the  changes  being 


MICROSCOPE  AND  ACCESSORIES 


\_CH.   I 


"Tube-length"  in 
Millimeters. 


In  the  following  tables  tube-length  b-d  of  the  diagram  greatly  preponderates, 
and  a  large  majority  of  unadjustable  objectives  are  corrected  for  a  thickness  of  cover- 
glass  falling  between  fifteen  and  twenty  hundredths  of  a  millimeter  (o.  15-0. 20  mm. ) . 
Length  in  Millimeters  and  Parts  included  in  the  '"Tube-Length"  by 

Various  Opticians. 
Pts.  included 
in  "Tube- 
length.  ' ' 
See  Diagram. 

fChas.  Baker,  London,  England 150  or  250  mm. 

The  Bausch  &  Lomb  Optical  Co. , 

Rochester,  N.  Y 160  or  216  mm. 

R.  &  J.  Beck,  London,  England 160  or  220  mm. 

Bezu,  Hausser  &  Cie,  Paris,  France 180  mm. 

Klonne  und  Miiller,  Berlin,  Germany 160  or  250  mm. 

Queen  &  Co.,  Incorporated,  Phila  ,  Pa 170  mm. 

Ross,  Ltd,  London,  England 160  or  254  mm. 

W.  und  H.  Seibert,  Wetzlar,  Germany 170  mm. 

Swift  &  Son,  London,  England 160  or  228  mm. 

Watson  &  Sons,   London,   England 160  or  250  mm. 

R.  Winkel,   Goettingen,  Germany 192  mm. 

[Carl  Zeiss,  Jena,  Germany 160  or  250 mm. 

f  Ernst  Leitz,  Wetzlar,  Germany 170  mm. 

|  Nachet  et  Fils,  Paris,  France 160  mm. 

j  Powell  &  Lealand,  London,  England 254mm. 

I  C.  Reichert,  Vienna,  Austria 160-180  mm. 

|  Spencer  Lens  Company,  Buffalo,  N.  Y 160  mm. 

[  W.  Wales,  New  York 254  mm. 

The  Gundlach  Opt.  Co.,  Rochester,  N.  Y.  254  mm. 

E.  Hartnack,  Potsdam,  Germany 160  mm. 

Dollond  &  Co.,  London,  England 165,  240  mm. 

Ve"rick  (Stiassnie)  Paris,  France 160-200  mm. 

P.  Wachter,  Berlin-Friedenau,  Germany  160  mm. 

J.  Zentmayer,  Philadelphia,  Pa, i6oor235tnm. 

Cover-Glass  for  Which  Non-Adjustable  Objectives  are  Corrected  by 

Various  Opticians. 

f  The  Bausch  &  Lomb  Optical  Co.,  Rochester,  N.  Y. 
J  Klonne  und  Miiller,  Berlin,  Germany, 
j  Queen  &  Co.,  Incorporated,  Philadelphia,  Pa. 
[The  Spencer  Lens  Co.,  Buffalo,  N.  Y. 
f  Ernst  Leitz,  Wetzlar,  Germany. 
•I  P.  Wachter,  Berlin-Friedenau,  Germany. 
I  R.  Winkel,  Goettingen,  Germany. 

(Chas.  Baker,  London,  England. 
R.  &  J.  Beck,  Ltd.,  London,  England. 
Gundlach  Optical  Co.,  Rochester,  N.  Y. 
W.  und  H.  Seibert,  Wetzlar,  Germany. 
E.  Hartnack,  Potsdam,  Germany. 
C.  Reichert,  Vienna,  Austria. 
Ross,  Ltd.,  London,  England. 
•  Verick  (Stiassnie),  Paris,  France. 
(  Carl  Zeiss,  Jena,  Germany. 

J.  Zentmayer,  Philadelphia,  Pa. 
J  Dollond  &  Co.,  London,  England. 
\  Nachet  et  Fils,  Paris,  France. 

Be"zu  Hausser  &  Cie,  Paris,  France, 
f  Powell  &  Lealand,  London,  England. 
\  Swift  &  Son,  London,  England. 
Watson  &  Sons,  London,  England. 
W.  Wales,  New  York. 


FIG.  24. 
Thickness  of 

o.  18  mm. 
0.17  mm. 

0.15  mm. 

0.15-0.18  mm.       \ 

0.15-0.20  mm 

0.12-0.17  mm 
0.10-015  mm. 
0.10-0.12  mm 
o.io  mm. 

0.20  mm. 
0.25  mm. 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


§  30.  Aperture  of  Objectives. — The  angular  aperture  or  angle 
of  aperture  of  an  objective  is  the  "angle  contained,  in  each  case,  be- 
tween the  most  diverging  of  the  rays  issuing  from  the  axial  point  of  an 
object  [/.  £.,  a  point  in  the  object  situated  on  the  optic  axis  of  the 
microscope],  that  can  enter  the  objective  and  take  part  in  the  formation 
of  an  image. ' '  (Carpenter) . 

in  the  direction  of  uniformity  and  in  general  in  the  direction  recommended  by  the 
writer  and  Mr.  Bausch  and  the  committee  of  the  American  Microscopical  Society. 
The  recommendations  of  the  committee,  published  in  the  Proceedings,  Vol.  XII., 
p.  250,  are  as  follows  : 

"Believing  in  the  desirability  of  a  uniform  tube-length  for  microscopes,  we 
unanimously  recommend  :  i.  That  the  parts  of  the  microscope  included  in  the 
tube-length  should  be  the  same  by  all  opticians,  and  that  the  parts  included  should 
be  those  between  the  upper  end  of  the  tube  where  the  ocular  is  inserted  and  the 
lower  end  of  the  tube  where  the  objective  is  inserted. 

2.  That  the  actual  extent  of  tube 
length  as  denned  in  section  i — Be,  for  the 
short  or  continental  tube,  160  mm.,  or  6.3 
inches,  and  216  mm.,  or  8^  inches,  for 
the  long  tube,  and  that  the  draw  tube  of 
the     microscope     possess    two     special 
marks  indicating  these  standard  lengths. 

3.  That  oculars  be  made  par-focal, 
and  that  the  par-focal  plane  be  coincident 
with  that  of  the  upper  end  of  the  tube. 

4.  That  the  mounting  of  all  object- 
ives of  6  mm.  (%  inch)  and  shorter  focus 
should  be  such  as  to  bring  the  optical 
center  of  the  objective  \%  inches  below 
the  shoulder,  and  that  all  objectives  be 
marked  with  the  tube-length  for  which 
they  are  corrected. 

5.  That  non-adjustable  objectives  be 
corrected  for  cover-glass  from  ^  to  T20°¥ 
mm.  (Iy^  to  T|^  inch)  in  thickness. 

These  recommendations  give  a  dis- 
tance of  10  inches  (254  mm.)  between  the 
par-focal  plane  of  the  ocular  and  the  op- 
tical center  of  the  objective  for  the  long 
tube,  and  are  essentially  in  accord  with 
the  actual  practice  of  opticians. 

At  the  request  of  the  committee,  a 
joint  conference  was  held  with  the  opti- 
cians belonging  to  the  Society  and  present  at  the  meeting.  They  expressed  their 
belief  in  the  entire  practicability  of  the  above  recommendations  and  a  willingness 
to  adopt  them." 

(Signed)  SIMON  H.  GAGE, 

A.  CLIFFORD  MERCER, 
CHARLES  E.  BARR. 


FIG.  25.  The  tube  of  a  microscope  with 
ocular  micrometer  and  nose  piece  in 
position  to  show  that  in  measuring 
tube-length  one  must  measure  from 
the  eye  lens  to  the  place  where  the  ob- 
jective is  attached.  (Zeiss1  Catalog.} 


1 6  MICROSCOPE  AND  ACCESSORIES  \CH.  I 

In  general  the  angle  increases  with  the  size  of  the  lenses  forming  the  objective 
and  the  shortness  of  the  equivalent  focal  distance  (\  15).  If  all  objectives  were 
dry  or  all  water  or  all  homogeneous  immersion  a  comparison  of  the  angular  aper- 
ture would  give  one  a  good  idea  of  the  relative  number  of  image  forming  rays 

FIG.  26.  Diagram  illustrating  the  angular  aperture  of 
a  microscopic  objective.  Only  the  front  lens  of  the  objective 
is  shown. 

Axis.     The  principal  optic  axis  of  the  objective. 

B  A,  B  C,  the  most  divergent  rays  that  can  enter  the 
objective,  they  mark  the  angular  aperture.  A  B  D  or  C  B 
D  half  the  angular  aperture.  This  is  designated  by  u  in 
making  Numerical  Aperture  computations.  See  the  table,  $  33. 

transmitted  by  different  objectives  ;  but  as  some  are  dry, 
others  water  and  still  others  homogeneous  immersion,  one 
can  see  at  a  glance  that,  other  things  being  equal,  the  dry 
objective  (Fig.  27)  receives  less  light  than  the  water  immersion,  and  the  water  im- 
mersion (Fig.  28)  less  than  the  homogeneous  immersion  (Fig.  29).  In  order  to 
render  comparison  accurate  between  different  kinds  of  objectives,  Professor 
Abbe  takes  into  consideration  the  rays  actually  passing  from  the  back  combi- 
nation of  the  objectives  to  form  the  real  image  ;  he  thus  takes  into  account  the 
medium  in  front  of  the  objective  as  well  as  the  angular  aperture.  The  term 
"Numerical  Aperture,"  (N.  A.}  was  introduced  by  Abbe  to  indicate  the  capacity 
of  an  optical  instrument  "for  receiving  rays  from  the  object  and  transmitting 
them  to  the  image. 

\  31.  Numerical  Aperture  (abbreviated  N.  A.),  as  now  employed  for  micro- 
scope objectives,  is  the  ratio  of  the  semi-diameter  of  the  emergent  pencil  to  the 
focal  length  of  the  lens.  Or  as  the  factors  are  more  readily  obtainable  it  is  sim- 
pler to  utilize  the  relationship  shown  in  the  La  Grange- Helmholtz- Abbe  formula, 
and  indicate  the  aperture  by  the  expression  :  N.  A.=«  sin  u.  In  this  formula  n 
is  the  index  of  refraction  of  the  medium  in  front  of  the  objective  (air,  water  or 
homogeneous  liquid) ,  and  sin  u  is  the  sine  of  half  the  angle  of  aperture  ( Fig.  26, 
DBA).  For  the  mathematical  discussion  showing  that  the  expressions 

semi-diameter  of  emergent  pencil  .  . 

— .      n  , -: 7-7? — ; —        —  =  n  sin  u,  the  student  is  referred  to  the  journal 

focal  length  of  the  lens 

of  the  Royal  Microscopical  Society ,  i88i,pp.  392-395,  1898,  p.  363. 

For  example,  take  three  objectives  each  of  3  mm.  equivalent  focus,  one  being 
a  dry,  one  a  water  immersion,  and  one  a  homogeneous  immersion.  Suppose  that 
the  dry  objective  has  an  angular  aperture  of  106°,  the  water  immersion  of  94°  and 
the  homogeneous  immersion  of  90°.  Simply  compared  as  to  their  angular  aper- 
ture, without  regard  to  the  medium  in  front  of  the  objective,  it  would  look  as  if 
the  dry  objective  would  actually  take  in  and  transmit  a  wider  pencil  of  light  than 
either  of  the  others.  However,  if  the  medium  in  front  of  the  objective  is  con- 
sidered, that  is  to  say,  if  the  numerical  instead  of  the  angular  apertures  are 
compared,  the  results  would  be  as  follows  :  Numerical  Aperture  of  a  dry  objective 
of  106°,  N.  A.=«  sin  u.  In  the  case  of  dry  objectives  the  medium  in  front  of  the 
objective  being  air,  the  index  of  refraction  is  unity,  whence  «— 1.  Half  the 
angular  aperture  is  ^  °=53°.  By  consulting  a  table  of  natural  sines  it  will  be 
found  that  the  sine  of  53°  is  0.799,  whence  N.  A.=«  or  1  X  sin  u  or  0.799—0.799.* 

*\  32.  Interpolation. — In  practice,  as  in  solving  problems  similar  to  those 
on  the  following  pages  and  those  in  refraction  if  one  cannot  find  a  sine  exactly 


CH.  /.] 


MICROSCOPE  AND  ACCESSORIES 


FIGS.  27-29  are  somewhat  modified  from 
Ellenberger,  and  are  introduced  to  illustrate 
the  relative  amount  of  utilized  light,  with  dry, 
water  immersion  and  homogeneous  immer- 

27  sion  objectives  of  the  same  equivalent  focus. 
The  point  from  which  the  rays  emanate  is  in 
air  in  each  case.     If  Canada  balsam  were  be- 
neath the  cover-glass  in  place  of  the  air  there 
would  be  practically  no  refraction  of  the  rays 
on  entering  the  cover  glass  (§  /£). 

FIG.  27.    Showing  the  course  of  the  rays 

28  passing  through  a  cover  glass  from  an  axial 
point  of  the  object,   and  the  number  that 
finally  enter  the  front  of  a  dry  objective. 

FIG.  28.     Rays  from  the  axial  point  of 
the  object  traversing  a  cover  of  the  same  thick- 
ness as  in  Fig.  27,  and  entering  the  front 
lens  of  a  water  immersion  objective. 
FIG.  29.     Rays  from  an  axial  point  of  the 

29  object  traversing  a  cover  glass  and  entering 
the  front   of  a     homogeneous    immersion 
objective. 

With  the  water  immersion  objective  the  medium  in  front  is 
water,  and  its  index  of  refraction  is  1.33,  whence  n=  1.33.  Half 
the  angular  aperture  is  -9^  °=  47°,  and  by  the  table  the  sine  of  47°  is 
found  to  be  0.731,  i.  e.,  sin  2^=0.731,  whence  N.  A.=n  or  i.33Xsin  u 
or  0.731=0.972. 


\\\\ 


SAll// 

j*-^^^g^ 


//C 


corresponding  to  a  given  angle  ;  or  if  one  has  an  angle  which  does  not  correspond 
to  any  sine  or  angle  given  in  the  table,  the  sine  or  angle  may  be  closely  approxi- 
mated by  the  method  of  interpolation,  as  follows  :  Find  the  sine  in  the  table  nearest 
the  sine  whose  angle  is  to  be  determined.  Get  the  difference  of  the  sines  of  the 
angles  greater  and  less  than  the  sine  whose  angle  is  to  be  determined.  That  will  give 
the  increase  of  sine  for  that  region  of  the  arc  for  15  minutes.  Divide  this  increase 
by  15  and  it  will  give  with  approximate  accuracy  the  increase  for  I  minute.  Now 
get  the  difference  between  the  sine  whose  angle  is  to  be  determined  and  the  sine 
just  below  it  in  value.  Divide  this  difference  by  the  amount  found  necessary  for 
an  increase  in  angle  of  i  minute  and  the  quotient  will  give  the  number  of  minutes 
the  sine  is  greater  than  the  next  lower  sine  whose  angle  is  known.  Add  this  num- 
ber of  minutes  to  the  angle  of  the  next  lower  sine  and  the  sum  will  represent  the 
desired  angle  of  the  sine.  Or  if  the  sine  whose  angle  is  to  be  found  is  nearer  in 
size  to  the  sine  just  greater,  proceed  exactly  as  before,  getting  the  difference  in  the 
sines,  but  subtract  the  number  of  minutes  of  difference  and  the  result  will  give  the 
angle  sought.  For  example  take  the  case  in  Section  97  where  the  sine  of  the 
angle  of  28°  54'  is  given  as  0.48327.  If  one  consults  the  table  the  nearest  sines 
found  are  0.48099,  the  sine  of  28°  45',  and  0.48461,  the  sine  of  29°.  Evidently 


1 8  MICROSCOPE  AND  ACCESSORIES  {_CH.  I 

With  the  oil  immersion  in  the  same  way  N.  A.=  n  sin  u  ;  n  or  the 
index  of  refraction  of  the  homogeneous  fluid  in  front  of  the  objective 
is  1.52,  and  the  semi-angle  of  aperture  is  ^-0—4.$°.  The  sine  of  45° 
is  0.707,  whence  N.  A.=n  or  152  x  sin  u  or  0.707=1.074. 

By  comparing  these  numerical  apertures  :  Dry  0.799,  water  0.972, 
homogeneous  immersion  1.074,  tne  same  idea  of  the  real  light  efficiency 
and  image  power  of  the  different  objectives  is  obtained,  as  in  the  graphic 
representations  shown  in  Figs.  27-29. 

If  one  knows  the  numerical  aperture  (N.  A.)  of  an  objective  the 
angular  aperture  is  readily  determined  from  the  formula  ;  and  one 
can  determine  the  equivalent  angles  of  objectives  used  in  different 
media  (i.  e.,  dry  or  immersion).  For  example,  suppose  each  of  three 
objectives  has  a  numerical  aperture  (N.  A.)  of  0.80,  what  is  the  an- 
gular aperture  of  each  ?  Using  the  formula  of  N.  A.=w  sin  u,  one  has 
N.  A.  =  0.80  for  all  the  objectives. 
For  the  dry  objective  n  =  i  (Refractive  index  of  air). 

"       water  immersion  objective  77=1.33  (Refractive  index  of  water). 
"        homogeneous  immersion  objective  n=  1.52  (Refractive   index 
of  homogeneous  liquid).     And  2  u  is  to  be  found  in  each  case. 

For  the  dry  objective,  substituting  the  known  values  the  formula 
becomes  0.80=  i  sin  u,  or  sin  u  =  0.80.  By  inspecting  the  table  of 
natural  sines  (3d  page  of  cover)  it  will  be  found  that  0.80  is  the  sine 
of  53  degrees  and  8  minutes.  As  this  is  half  the  angle  the  entire 
angular  aperture  of  the  dry  objective  must  be  53°  8'x  2  =  106°  16'. 

For  the  water  immersion  objective,  substituting  the  known  values 

in  the  formula  as  before  :  0.80  =  1.33  sin  u,  or  sin  u=    —  =  0.6015. 

*  *3O 

Consulting  the  table  of  sines  as  before,  it  will  be  found  that  0.6015  is 
the  sine  of  36°  59'  whence  the  angular  aperture  (water  angle)  is  36° 
59'X2  =  73058'. 

For  the  homogeneous  immersion  objective,  substituting  the  known 
values,  the  formula   becomes:    0.80  =  1.52    sin   u   whence   sin  *u  = 
0.80 


1-52 


=  0.5263.     And  by  consulting  the  table  of  sines  it  will  be  found 


then  the  angle  sought  must  lie  between  28°  45',  and  29°.  If  the  difference  between 
0.48481  and  0.48099  be  obtained,  0.48481  — 0.48099  =  0.00382,  and  if  this  increase  for 
15'  be  divided  by  15  it  will  give  the  increase  for  i  minute  ;  0.00382  -f-  15  =0.000254. 
Now  the  difference  between  the  sine  whose  angle  is  to  be  found  and  the  next 
lower  sine  is  0.48327—0.48099  =  0.00228.  If  this  difference  be  divided  by  the 
amount  found  necessary  for  i  minute  it  will  give  the  total  minutes  above  28°  45'; 
o. 002 28  -5-  0.000254  =  9.  That  is,  the  angle  sought  is  9  minutes  greater  than 


\_CH.  I 


MICROSCOPE  AND  ACCESSORIES 


that  this  is  the  sine  of  31°  45^'  whence  2  u  or  the  entire  angle  (balsam 
or  oil  angle)  is  63°  31'. 

That  is,  three  objectives  of  equal  resolving  powers,  each  with  a 
numerical  aperture  of  0.80  would  have  an  angular  aperture  of  106°  16' 
in  air,  73°  58'  in  water  and  63°  31'  in  homogeneous  immersion  liquid. 

For  the  apparatus  and  method  of  determining  aperture,  see 
appendix. 

§  33.  Table  of  a  Group  of  Objectives  with  the  Numerical 
Aperture  (N.  A)  and  the  method  of  obtaining  it.  Half  the  angular 
aperture  is  designated  by  u  and  the  index  of  refraction  of  the  medium  in 
front  of  the  objective  by  n.  For  dry  objectives  this  is  air  and  n  =  i,  fot 
water  immersions  n  =  f.jj,  and  for  homogeneous  immersions  n  =  1. 
(For  a  table  of  natural  sines,  see  third  page  of  cover. ) 


OBJECTIVE. 

!P 

Index  of 

NATURAL  SINE      Refraction 
of  half  the  angular   of  the  medi- 
.       ._                 um  in  front 
aperture              oftheobjec- 
(sinu.  )                   tive  (n). 

NUMERICAL  APERTURE 
(N.  A.)  =/zsin  u 

25  mm. 

(Dry.) 

20° 

Sin  —  =  0.1736   j     n  =  i        N.A.= 

i  Xo.  1736  =0.173 

25  mm. 

(Dry.) 

40° 

40 
Sin  -^—  =  0.3420        »  —  i 

N.A.= 

i  X  0.3420  =0.342 

12^2  mm. 
(Dry.) 

42° 

Sin  ^-  =  0.3584        n=l 

N.A.= 

i  Xo.  3583  =0.358 

12^  mm. 
(Dry.) 

100° 

Sin  ^°  =  0.7660        «  =  i        N.A.= 

i  X  0.7660  =  0.766 

6  mm. 

(Dry.) 

75° 

Sin—  -  =  0.6087        n  =  i 

N.A.= 

i  Xo.  6087  =0.609 

6  mm. 
(Dry.) 

136° 

Sin  -*r  =  0.9272        n  =  i 

N.  A.= 

1X0.9272=0.927 

3  mm. 
(Dry.) 

115° 

Sin  ^=0.8434        n  =  l 

N.  A.= 

1X0.8434  =  0.843 

3  mm. 
(Dry.) 

163° 

Sin  ^  =  0.9890        n=^1 

N.  A.= 

1X0.9890  =  0.989 

2  mm. 
Water. 
Immersion. 

96°  1  2' 

N.  A.=i.33X  0.7443  =  0.99 

oin                 —  0.7443        '*        L'6o 

2  mm. 
Homogeneous 
Immersion. 

no°38/ 

-inIIO°38/       n^'          *«!« 

C5in                —  0.0^^3      '*       ^-o-^ 

2  mm. 
Homogeneous 
Immersion. 

I34°io/ 

• 

N.  A.= 

1.52X0.9210  =  1.40 

20  MICROSCOPE  AND  ACCESSORIES  \_CH.  I 

§  34.  Significance  of  Aperture. — As  to  the  real  significance  of 
aperture  in  microscopic  objectives,  it  is  now  an  accepted  doctrine  that — 
the  corrections  in  spherical  and  chromatic  aberration  being  the  same — 
(i)  Objectives  vary  directly  as  their  numerical  aperture  in  their  ability 
to  define  or  make  clearly  visible  minute  details  (resolving  power).  For 
example  an  objective  of  4  mm.  equivalent  focus  and  a  numerical  aper- 
ture of  0.50  would  define  or  resolve  only  half  as  many  lines  to  the 
millimeter  or  inch  as  a  similar  objective  of  i.oo  N.A.  So  also  an 
objective  of  2  mm.  focus  and  1.40  N.A.  would  resolve  only  twice  as 
many  lines  to  the  millimeter  as  a  4  mm.  objective  of  0.70  N.A.  Thus 
it  is  seen  that  defining  power  is  not  a  result  of  magnification  but  of 
aperture,  otherwise  the  2  mm.  objective  would  resolve  far  more  than 
twice  as  many  lines  as  the  4  mm.  objective. 

Taking  the  results  of  the  researches  of  Abbe  as  a  guide  to  visibility 
with  the  microscope,  one  has  the  general  formula  2\xN.A.  That  is 
twice  the  number  of  wave  lengths  of  the  light  used  multiplied  by  the 
numerical  aperture  of  the  objective.  From  this  general  statement  it  will 
be  seen  that  the  shorter  the  wave  lengths  of  the  light,  the  more  there 
will  be  in  an  inch  or  centimeter  and  therefore  the  greater  the  number  of 
lines  visible  in  a  given  space.  That  is  the  kind  of  light  used  is  one  ele- 
ment and  the  objective  the  other  in  determining  the  number  of  lines 
visible  under  the  microscope. 

Following  Mr.  E.  M.  Nelson  (Jour.  Roy.  Micr.  Soc.,  1893,  P-  J5) 
it  is  believed  that  not  more  than  ^ths  of  the  numerical  aperture  of  an 
objective  is  really  available  for  microscopic  study,  with  a  central,  solid 
cone  of  light.  To  determine  the  number  of  lines  visible  in  a  given  space 
with  a  given  light  the  formula  would  become  2\  X  3/^ths  N.A.  — 3/2/lN.  A. 
To  determine  the  working-resolving  power  of  any  objective  it  is  only 
necessary  to  know  the  number  of  light  waves  in  a  given  space,  say  an 
inch  or  a  centimeter  and  to  multiply  this  number  by  3/2  N.  A.  For 
example  suppose  one  uses  ordinary  daylight  and  assumes  the  average 
wavelength  is  1/46666  in.,  then  there  must  be  46,666  per  inch  and 
46,666x3/2  =  70,000  approximately.  If  the  N.  A.  is  i,  then  the 
objective  will  resolve  or  make  visible  70,000  lines  to  the  inch,  or  ap- 
proximately 28,000  to  the  centimeter.  If  blue  light  were  used,  the 
number  would  be  32,000  per  centimeter,  or  80,000  per  inch.  It  will 
be  seen  that  the  number  of  lines  here  given  is  smaller  than  that  in  the 
table  of  Carpenter-Dallinger,  because  in  the  latter  the  full  aperture 
is  supposed  to  be  employed  and  the  light  is  of  the  greatest  available 
obliquity. 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  21 

(2)  The  illuminating  power  of  an  objective  of  a  given  focus  is 
found  to  vary  directly  as  the  square  of  the  numerical  aperture  (N.  A.  )2. 
Thus  if  two  4  mm.  objectives  of  N.A.  0.20  and  N.A.  0.40  were  compared 
as  to  their  illuminating  power  it  would  be  found  from  the  above  that 
they  would  vary  as  o.2o2:o.402=  0.0400:0.1600  or  1:4.     That  is  the 
objective  of  0.20  N.A.  would  have  but  ^th  the  illuminating  power  of 
the  one  of  0.40  N.A. 

(3)  The  penetrating  power,  that  is  the  power  to  see  more  than  one 

plane,  is  found  to  vary  as  the  reciprocal  of  the  numerical  aperture 

so  that  in  an  objective  of  a  given  focus  the  greater  the  aperture  the 
less  the  penetrating  power. 

Of  course  when  equivalent  focus  and  numerical  aperture  both  differ 
the  problem  becomes  more  complex. 

While  all  microscopists  are  agreed  that  the  fineness  of  detail  which 
can  be  seen  depends  directly  on  the  numerical  aperture  of  the  objective 
used,  the  general  theory  of  microscopic  vision  has  two  interpretations  : 

(A.)  That  it  is  as  with  the  unaided  eye,  the  telescope  and  the 
photographic  camera.  This  is  the  original  view  and  the  one  which  many 
are  favoring  at  the  present  day  (see  Mercer,  Proceedings  of  the  Amer. 
Micr.  Soc.  1896,  pp.  321-396). 

(B)  The  other  view  originated  with  Professor  Abbe,  and  in  the 
words  of  Carpenter-Dallinger,  pp.  62,  43  :  ''What  this  is  becomes  ex- 
plicable by  the  researches  of  Abbe.  It  is  demonstrated  that  micro- 
scopic vision  is  sui generis.  There  is  and  can  be,  no  comparison  between 
microscopic  and  macroscopic  vision.  The  images  of  minute  objects 
are  not  delineated  microscopically  by  means  of  the  ordinary  laws  of 
refraction  ;  they  are  not  dioptrical  results,  but  depend  entirely  on  the 
laws  of  diffraction.  These  come  within  the  scope  of  and  demonstrate 
the  undulatory  theory  of  light,  and  involve  a  characteristic  change 
which  material  particles  or  fine  structural  details,  in  proportion  to  their 
minuteness,  effect  in  transmitted  rays  of  light.  The  change  consists 
generally  in  the  breaking  up  of  an  incident  ray  into  a  group  of  rays 
with  large  angular  dispersion  within  the  range  of  which  periodic  alter- 
nations of  dark  and  light  occur. ' ' 

For  a  consideration  of  the  aperture  question,  its  history  and  sig- 
nificance, see  J.  D.  Cox,  Proc.  Amer.  Micr.  Soc.,  1884,  pp.  5-39  ; 
Jour.  Roy.  Micr.  Soc.,  1881,  pp.  303,  348,  365,  388  ;  1882,  pp.  300, 
460  ;  1883,  p.  790  ;  1884,  p.  20  ;  1896,  p.  681  ;  1897,  p.  71  ;  1898,  pp. 
354,  362,  592  ;  Mercer,  Proceedings  Amer.  Micr.  Soc.,  1896,  pp.  321- 


22 


MICROSCOPE  AND  ACCESSORIES 


[CH.  I 


396  ;  Lewis  Wright,  Philos.  Mag.,  June,  1898,  pp.  480-503  ;  Carpen- 
ter-Dallinger,  Chapter  II ;  Nelson,  Jour.  Quekett  Micr.  Club,  VI,  pp. 
14-38. 

THE   OCULAR 

g  35.  A  Microscopic  Ocular  or  Eye-Piece  consists  of  one  or  more  converging 
lenses  or  lens  systems,  the  combined  action  of  which  is,  like  that  of  a  simple 
microscope,  to  magnify  the  real  image  formed  by  the  objective. 

FIG.  30.  Sectional  view  of  a  Huygenian  ocular  to  show 
the  formation  of  the  Eye-Point. 

Axis  .Optic  axis  of  the  ocular.  D.  Diaphragm  of  the 
ocular.  E.  L.  Eye-Lens.  F.  L.  Field-Lens. 

E.  P.  Eye-point.  As  seen  in  section,  it  appears  some- 
thing like  an  hour-glass.  When  seen  as  looking  into  the 
ocular,  i.  <?.,  in  transection,  it  appears  as  a  circle  of  light.  It 
is  at  the  point  where  the  most  rays  cross. 

Depending  upon  the  relation  and  action  of  the  different 
lenses  forming  oculars,  they  are  divided  into  two  great 
groups,  negative  and.  positive. 

36.  Negative  Oculars  are  those  in  which  the  real,  inverted  image  is  formed 
within  the  ocular,  the  lower  or  field-lens  serving  to  collect  the  image-forming  rays 
somewhat,  so  that  the  real  image  is  smaller  than  as  if  the  field-lens  were  absent 
(Fig.  21 ).  As  the  field-lens  of  the  ocular  aids  in  the  formation  of  the  real  image 
it  is  considered  by  some  to  form  a  part  of  the  objective  rather  than  of  the  ocular. 
The  upper  or  eye-lens  of  the  ocular  magnifies  the  real  image. 

§  37.  Positive  Oculars  are  those  in  which  the  real,  inverted  image  of  the 
object  is  formed  outside  the  ocular,  and  the  entire  system  of  ocular  lenses  magnifies 
the  real  image  like  a  simple  microscope  (Fig.  16). 

Positive  and  negative  oculars  may  be  readily  distinguished,  as  a  positive  ocular 
may  be  used  as  a  simple  microscope,  while  a  negative  ocular  cannot  be  so  used 
when  its  field-lens  is  in  the  natural  position  toward  the  object.  By  turning  the 
eye-lens  toward  the  object  and  looking  into  the  field-lens  an  image  may  be  seen, 
however. 

In  works  and  catalogs  concerning  the  microscope  and  microscopic  apparatus, 
and  in  articles  upon  the  microscope  in  periodicals,  various  forms  of  oculars  or  eye- 
pieces are  so  frequently  mentioned,  without  explanation  or  definition,  that  it 
seems  worth  while  to  give  a  list,  with  the  French  and  German  equivalents,  and  a 
brief  statement  of  their  character. 

Achromatic  Ocular  ;  Fr.  Oculaire  achromatique  ;  Ger.  achromatisches  Okular. 
Oculars  in  which  chromatic  aberration  is  wholly  or  nearly  eliminated. — Aplanatic 
Ocular  ;  Fr.  Oculaire  aplanatique  ;  Ger.  aplanatisches  Okular  (see  \  21). — Binocu- 
lar, stereoscopic  Ocular  ;  Fr.  Oculaire  binoculaire  stereoscopique  ;  Ger.  stereosko- 
pisches  Doppel-Okular.  An  ocular  consisting  of  two  oculars  about  as  far  apart  as 
the  two  eyes.  These  are  connected  with  a  single  tube  which  fits  a  monocular  mi- 
croscope. By  an  arrangement  of  prisms  the  image  forming  rays  are  divided,  half 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  23 

being  sent  to  each  eye.  The  most  satisfactory  form  was  worked  out  by  Tolles  and 
is  constructed  on  true  stereotomic  principles,  both  fields  being  equally  illuminated. 
His  ocular  is  also  erecting. — Campani^s  Ocular  (see  Huygenian  Ocular). — Com- 
pound Ocular  ;  Fr.  Oculaire  compose  ;  Ger.  zusammengesetztes  Okular.  An  ocu- 
lar of  two  or  more  lenses,  e.  g.,  the  Huygenian  (see  Fig.  30). — Continental  Ocular. 
An  ocular  mounted  in  a  tube  of  uniform  diameter  as  in  Fig.  ^i.—Deep  Ocular, 
see  high  ocular. — Erecting  Ocular  ;  Fr.  Oculaire  redresseur  ;  Ger.  bildumkeh- 
rendes  Okular.  An  ocular  with  which  an  erecting  prism  is  connected  so  that  the 
image  is  erect  as  with  the  simple  microscope.  Such  oculars  are  most  common  on 
dissecting  microscopes. — Filar  micrometer  Ocular;  Screw  m.  o.,  Cobweb  m.  o., 
Ger.  Okular-Schraubenmikrometer.  A  modification  of  Ramsden's  Telescopic  Cob- 
web micrometer  ocular.  —  Goniometer  Ocular  ;  Fr.  Oculaire  a  goniometre  ;  Ger. 
Goniometer-Okular.  An  ocular  with  goniometer  for  measuring  the  angles  of  minute 
crystals. — High  Ocular,  sometimes  called  a  deep  ocular.  One  that  magnifies 
the  real  image  considerably,  i.  e.,  10  to  20  fold. — Huygenian  Ocular,  Huygens'  O., 
Campani's  O.,  Airy's  O.;  Fr.  Oculaire  d'Huygens,  o.  de  Campani  ;  Ger.  Huy- 
gens'sches  Okular,  Campaniches  Okular,  see  \  38. — Index  Ocular ;  Ger.  Spitzen- 
O.  An  ocular  with  a  minute  pointer  or  two  pointers  at  the  level  of  the  real  image. 
The  points  are  movable  and  serve  for  indicators  and  also,  although  not  satisfac- 
torily, for  micrometry. — Kellner's  Ocular,  see  orthoscopic  ocular — Low  ocular, 
also  called  shallow  ocular.  An  ocular  which  magnifies  the  real  image  only  moder- 
ately, i.  e. ,  2  to  8  fold. — Micrometer  or  micrometric  Ocular  ;  Fr.  Oculaire  microme- 
trique  ou  a  micrometre  ;  Ger.  Mikrometer-Okular,  Mess  Okular,  Beneches  O., 
Jackson  m.  o.,  see  \  41. — Microscopic  Ocular  ;  Fr.  Oculaire  microscopique  ;  Ger.  mi- 
kroskopisches  Okular.  An  ocular  for  the  microscope  instead  of  one  for  a  telescope. 
— Negative  Ocular,  see  \  36. — Nelson's  screw-micrometer  ocular.  A  modification  of 
the  Ramsden's  screw  or  cob-web  micrometer  in  which  positive  compensating  ocu- 
lars may  be  used. — Orthoscopic  Oculars;  also  called  Kellner's  Ocular  ;  Fr.  Ocu- 
laire orthoscopique  ;  Ger.  Kellner'sches  oder  orthoskopisches  Okular.  An  ocular 
with  an  eye-lens  like  one  of  the  combinations  of  an  objective  (Figs.  22,  23)  and  a 
double  convex  field  lens.  The  field-lens  is  in  the  focus  of  the  eye-lens  and  there 
is  no  diaphragm  present.  The  field  is  large  and  flat. — Par-focal  Oculars,  a  series 
of  oculars  so  arranged  that  the  microscope  remains  in  focus  when  the  oculars  are 
interchanged  (Pennock,  Micr.  Bulletin,  vol.  iii,  p.  9,  31). — Periscopic  Ocular  ;  Fr. 
Oculaire  periscopique  ;  Ger.  periskopisches  Okular.  A  positive  ocular  devised  by 
Gundlach.  It  consists  of  a  double  convex  field-lens  and  a  triplet  eye-lens.  It 
gives  a  large,  flat  field. — Positive  Ocular,  see  \  37. — Projection  Ocular ;  Fr.  Ocu- 
laire de  projection  ;  Ger.  Projections-Okular,  see  \  40. — Ramsden's  Ocular ;  Fr. 
Oculaire  de  Ramsden  ;  Ger.  Ramsden'sches  Okular.  A  positive  ocular  devised  by 
Ramsden.  It  consists  of  two  plano-convex  lenses  placed  close  together  with  the 
convex  surfaces  facing  each  other.  Only  the  central  part  of  the  field  is  clear. 
Searching  Ocular  ;  Fr.  Oculaire  d'orientation ;  Ger.  Sucher-Okular,  see  §  39, 
Shallow  Ocular,  see  low  ocular. — Solid  Ocular,  holosteric  O. ;  Fr.  Oculaire  holo- 
stere  ;  Ger.  holosterisches  Okular,  Vollglass-Okular.  A  negative  eye-piece  de- 
vised by  Tolles.  It  consists  of  a  solid  piece  of  glass  with  a  moderate  curvature  at 
one  end  for  a  field-lens,  and  the  other  end  with  a  much  greater  curvature  for  an 
eye-lens.  For  a  diaphragm,  a  groove  is  cut  at  the  proper  level  and  filled  with 
black  pigment.  It  is  especially  excellent  where  a  high  ocular  is  desired. — Spectral 


24  MICROSCOPE  AND  ACCESSORIES  {CH.  I 

or  spectroscopic  Ocular  ;  Fr.  Oculaire  spectroscopique  ;  Ger,  Spectral-Okular,  see 
Microspectroscope,  Ch.  VI. — Statiroscopic  Ocular  ;  Fr.  Oculaire  Stauroscopique. 
Ger.  Stauroskop-Okular.  An  ocular  with  a  Bertrand's  quartz  plate  for  mineralog- 
ical  purposes. —  Working  Ocular;  Fr.  Oculaire  de  travail;  Ger.  Arbeits-Okular, 
see  §  39. 

g  38.  Huygenian  Ocular — A  negative  ocular  designed  by  Huygens  for  the 
telescope,  but  adapted  also  to  the  microscope.  It  is  the  one  now  most  commonly 
employed.  It  consists  of  a  field-lens  or  collective  (Fig.  30),  aiding  the  objective 
in  forming  the  real  image,  and  an  eye-lens  which  magnifies  the  real  image.  While 


Ocular  lo.  2 


FIG.  31.  Compensating  Oculars  of  Zeiss,  with  section  removed  to  show  the  con- 
struction. The  line  A- A  is  at  the  level  of  the  upper  end  of  the  tube  of  the  micro- 
scope while  B-B  represents  the  lower  focal  points.  It  will  be  seen  that  the  mount- 
ing is  so  arranged  that  the  lower  focal  points  in  all  are  in  the  same  plane  and 
therefore  the  microscope  remains  in  focus  upon  changing  oculars.  (  The  oculars  are 
par-focal}.  The  lower  oculars,  2,  4  and  6  are  negative,  and  the  higher  ones,  8,  12, 
18,  are  positive.  The  numbers  2,  4,  6,  8,  12,  18,  indicate  the  magnification  of  the 
ocular.  From  Zeiss'  Catalog. ) 

the  field-lens  aids- the  objective  in  the  formation  of  the  real,  inverted  image,  and 
increases  the  field  of  view,  it  also  combines  with  the  eye-lens  in  rendering  the 
image  achromatic.  (See  $46). 

$  39.  Compensating  Oculars. — These  are  oculars  specially  constructed  for 
use  with  the  apochromatic  objectives.  They  compensate  for  aberrations  outside 
the  axis  which  could  not  be  so  readily  eliminated  in  the  objective  itself.  An  ocu- 
lar of  this  kind,  magnifying  but  twice,  is  made  for  use  with  high  powers,  for  the 
sake  of  the  large  field  in  finding  objects  ;  it  is  called  a  searching  ocular ;  those 
ordinarily  used  for  observation  are  in  contradistinction  called  working  oculars. 
Part  of  the  compensating  oculars  are  positive  and  part  negative.  (Fig.  31. ) 

\  40.  Projection  Oculars.— These  are  oculars  especially  designed  for  project- 
ing a  microscopic  image  on  the  screen  for  class  demonstrations,  or  for  photo- 
graphing with  the  microscope.  While  they  are  specially  adapted  for  use  with 
apochromatic  objectives,  they  may  also  be  used,  with  ordinary  achromatic 
objectives  of  large  numerical  aperture. 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


FIG.  32.  Projection  Oculars  with  section  re- 
moved to  show  the  construction.  Below  are 
shown  the  upper  ends  with  graduated  circle  to 
indicate  the  amount  of  rotation  found  necessary 
to  focus  the  diaphragm  on  the  screen.  No.  2, 
No.  4.  The  numbers  indicate  the  amount  the 
ocular  magnifies  the  image  formed  by  the 
objective  as  with  the  compensation  oculars. 
(Zeis?  Catalog.} 

|  41.  Micrometer  Ocular. — This  is  an 
ocular  connected  with  an  ocular  micrometer. 
The  micrometer  may  be  removable,  or  it  may 
be  permanently  in  connection  with  the  ocular, 
and  arranged  with  a  spring  and  screw,  by  which 
it  may  be  moved  back  and  forth  across  the 
field.  (SeeCh.  IV.) 


No.  2. 


FIG.  33  FIG.  34 

FIGS.  33-34-  Ocular  Micrometer  and  movable  scale.  Fig.  33  is  a  side  view  of 
the  ocular  while  Fig.  34  gives  a  sectional  end  view,  and  shows  the  ocular  micrometer 
in  position.  In  both  the  screw  which  moves  the  micrometer  is  shown  at  the  left. 
( From  Bausch  &  Lomb  Opt.  Co. ) 

I  42.     Spectral  or  Spectroscopic  Ocular.— (See  Micro-Spectroscope,  Ch.  VI). 


DESIGNATION   OF   OCULARS 

\  43.  Equivalent  Focus. — As  with  objectives,  some  opticians  designate  the 
oculars  by  their  equivalent  focus  ( \  15 ).  With  this  method  the  power  of  the  ocular, 
as  with  objectives,  varies  inversely  as  the  equivalent  focal  length,  and  therefore 
the  greater  the  equivalent  focal  length  the  less  the  magnification.  This  seems  as 
desirable  a  mode  for  oculars  as  for  objectives  and  is  coming  more  and  more  into 
use  by  the  most  progressive  opticians.  It  is  the  method  of  designation  advo- 
cated by  Dr.  R.  H.  Ward  for  many  years,  and  was  recommended  by  the  committee 
of  the  American  Microscopical  Society,  (Proc.  Amer.  Micr.  Soc.,  1883,  p.  175,  1884, 
p.  228). 


26 


MICROSCOPE  AND  ACCESSORIES 


\_CH.I 


FIG.  35.  Ocular  Screw- Micrometer  with 
compensation  ocular  6.  The  upper  figure 
shows  a  sectional  view  of  the  ocular  and  the 
screw  for  moving  the  micrometer  at  the  right. 
At  the  left  is  shown  a  clamping  screw  to 
fasten  the  ocular  to  the  upper  part  of  the  mi- 
croscope tube.  Below  is  a  face  view,  showing 
the  graduation  on  the  wheel.  An  ocular 
micrometer  like  this  is  in  general  like  the 
cob-web  micrometer  and  may  be  used  for 
measuring  objects  of  varying  sizes  very  accu- 
rately. With  the  ordinary  ocular  micrometer 
very  small  objects  frequently  fill  but  a  part  of 
an  interval  of  the  micrometer,  but  with  this 
the  movable  cross  lines  traverse  the  object  (or 
rather  its  real  image]  regardless  of  the  minute- 
ness of  the  object.  (Zeiss^  Catalog}. 

\  44.  Numbering  and  Lettering. — Oculars  like  objectives  may  be  numbered  or 
lettered  arbitrarily.  When  so  designated,  the  smaller  the  number,  or  the  earlier 
the  letter  in  the  alphabet,  the  lower  the  power  of  the  ocular. 

$  45.  Magnification. — The  compensating  oculars  are  marked  with  the  amount 
they  magnify  the  real  image.  Thus  an  ocular  marked  X  4,  indicates  that  the  real 
image  of  the  objective  is  magnified  four  fold  by  the  ocular. 

The  projection  oculars  are  designated  simply  by  the  amount  they  multiply  the 
real  image  of  the  objective.  Thus  for  the  short  or  160  mm.  tube-length  they  are, 
X2,  X4  ;  and  for  the  long  or  250  mm.  tube,  they  are  X3  and  X6.  That  is,  the 
final  image  on  the  screen  or  the  ground  glass  of  the  photographic  camera  will  be 
2,  3,  4,  or  6  times  greater  than  it  would  be  if  no  ocular  were  used.  See  Ch.  VIII. 
§  46.  Standard  Size  Oculars. — The  Royal  Microscopical  Society  of  London 
took  a  very  important  step  (Dec.  20,  1899)  in  establishing  standard  sizes  for  ocu- 
lars and  sub-stage  condensers.  To  quote  from  the  Journal  of  the  Royal  Micro- 
scopical Society  for  1900,  p.  147  : 

Resolved,  "That  the  standard  size  for  the  inside  diameter  of  the  substage  fit- 
ting be  1.527  in.  =38. 786  mm.  That  the  gauges  for  standardizing  eye-pieces  be 
the  internal  diameters  of  the  draw-tubes,  the  tightness  of  the  fit  being  left  to  the 
discretion  of  the  manufacturers. ' ' 

The  sizes  for  oculars  are  four  in  number,  I  and  2  being  most  common. 
( i )  0.9173  inch  =  23.300  mm.     This  is  the  Continental  size. 

This  is  the  size  used  by  the  English  Opticians 

for  student  and  small  microscopes. 
Medium  size  binoculars  (English.) 
Long  tube  binoculars. 
For  the  history  of  the  Huygenian  Ocular,  and  a  discussion  of  formulae  for  its 
construction,  see  Nelson,  J.  R.  M.  S.,  1900,  p.  162-169. 

EXPERIMENTS 

§  47.     Putting  an  Objective  in  Position  and  Removing  it. — 
Elevate  the  tube  of  the  microscope  by  means  of  the  coarse  adjustment 


(2)   1.04      inch  =  26.416  mm. 


(3) 
(4) 


1.27 
1.41 


inch  =32.258  mm. 
=  35.814  mm. 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  27 

(frontispiece)  so  that  there  may  be  plenty  of  room  between  its  lower 
end  and  the  stage.  Grasp  the  objective  lightly  near  its  lower  end  with 
two  fingers  of  the  left  hand,  and  hold  it  against  the  nut  at  the  lower 
end  of  the  tube.  With  two  fingers  of  the  right  hand  take  hold  of  the 
milled  ring  near  the  back  or  upper  end  of  the  objective  and  screw  it 
into  the  tube  of  the  microscope.  Reverse  this  operation  for  removing 
the  objective.  By  following  this  method  the  danger  of  dropping  the 
objective  will  be  avoided. 

§  48.  Putting  an  Ocular  in  Position  and  Removing  it. — Ele- 
vate the  body  of  the  microscope  with  the  coarse  adjustment  so  that  the 
objective  will  be  2  cm.  or  more  from  the  object — grasp  the  ocular  by 
the  milled  ring  next  the  eye-lens  (Fig.  21),  and  the  coarse  adjustment 
or  the  tube  of  the  microscope  and  gently  force  the  ocular  into  position. 
In  removing  the  ocular,  reverse  the  operation.  If  the  above  precau- 
tions are  not  taken,  and  the  oculars  fit  snugly,  there  is  danger  in  in- 
serting them  of  forcing  the  tube  of  the  microscope  downward  and  the 
objective  upon  the  object. 

§  49.  Putting  an  Object  under  the  Microscope. — This  is  so 
placing  an  object  under  the  simple  microscope,  or  on  the  stage  of  the 
compound  microscope,  that  it  will  be  in  the  field  of  view  when  the 
microscope  is  in  focus  (§  50). 

With  low  powers,  it  is  not  difficult  to  get  an  object  under  the 
microscope.  The  difficulty  increases,  however,  with  the  power  of  the 
microscope  and  the  smallness  of  the  object.  It  is  usually  necessary  to 
move  the  object  in  various  directions  while  looking  into  the  micro- 
scope, in  order  to  get  it  into  the  field.  Time  is  usually  saved  by  get- 
ting the  object  in  the  center  of  the  field  with  a  low  objective  before 
putting  the  high  objective  in  position.  This  is  greatly  facilitated  by 
using  a  nose-piece,  or  revolver.  (See  Figs.  36-363,  and  the  pictures  of 
microscopes,  Ch.  II.) 


FIG.  36.    Triple  nose-piece  or  revol-  FIG.  363.       Triple  nose-piece  or  re- 

ver for  quickly  changing  objectives.  (  The      volver  for  quickly  changing  objectives. 
Spencer  Lens  Co. )  (  The  Bausch  &  Lomb  Optical  Co. ) 


28  MICROSCOPE  AND  ACCESSORIES  [CH.  I 

§  50.  Field  or  Field  of  View  of  a  Microscope. — This  is  the 
area  visible  through  a  microscope  when  it  is  in  focus.  When  properly 
lighted  and  there  is  no  object  under  the  microscope,  the  field  appears 
as  a  circle  of  light.  When  examining  an  object  it  appears  within  the 
light  circle,  and  by  moving  the  object,  if  it  is  of  sufficient  size,  differ- 
ent parts  are  brought  successively  into  the  field  of  view. 

In  general,  the  greater  the  magnification  of  the  entire  microscope, 
whether  the  magnification  is  produced  mainly  by  the  objective,  the 
ocular,  or  by  increasing  the  tube  length,  or  by  a  combination  of  all 
three  (see  Ch.  IV,  under  magnification),  the  smaller  is  the  field. 

The  size  of  the  field  is  also  dependent,  in  part,  without  regard  to 
magnification,  upon  the  size  of  the  opening  in  the  ocular  diaphragm. 
Some  oculars,  as  the  orthoscopic  and  periscopic,  are  so  constructed  as 
to  eliminate  the  ocular  diaphragm,  and  in  consequence,  although  this 
is  not  the  sole  cause,  the  field  is  considerably  increased.  The  exact 
size  of  the  field  may  be  read  off  directly  by  putting  a  stage  micrometer 
under  the  microscope  and  noting  the  number  of  spaces  required  to 
measure  the  diameter  of  the  light  circle. 


4  5 


17 


85    mm 


FIG.  37.  Figures  showing  approximately  the  actual  size  of  the  field  with  ob- 
jectives 0/85  mm.,  45  mm.,  77  mm.,  3  mm.,  and  2  mm.,  equivalent  focus,  and 
ocular  of  3jY2  mm.,  equivalent  focus  in  each  case.  This  figure  shows  graphically 
what  is  also  very  clearly  indicated  in  the  table  ( \  52}. 

§  51.  The  size  of  the  field  of  the  microscope  as  projected  into  the 
field  of  vision  of  the  normal  human  eye  (i.  e.,  the  virtual  image)  may 
be  determined  by  the  use  of  the  camera  lucida  with  the  drawing  surface 
placed  at  the  standard  distance  of  250  millimeters  (Ch.  IV.) 

§  52.  Table  showing  the  actual  size  in  millimeters  of  the  field  of  a 
group  of  commonly  used  objectives  and  oculars.  Compare  with  the  graphic 
representation  in  Fig.  37.  See  also  §  50. 


CH.r\ 


MICROSCOPE  AND  ACCESSORIES 


29 


Equivalent    i  Diameter 
**»*•$*    !   of  Field 
Objective         in  mm. 

Equivalent 
Focus  of 
Ocular 

Kind   of 
Ocular 

15-4 
85  mm              j       10.6 

37  X  mm. 
25         " 

Huygenian 

!         8'3 

I2tf       "' 

7-o 
45  mm  5.0 
4.0 

37  yz  mm. 
i2>£     " 

Huygenian 

3-o 
17  mm.                     2.0 

37>£  mm. 
25 

Huygenian 

1.6 

J«  •• 

5-7 

N.  A.  =0.25      J;4 

0.97 

180      mm. 
45        " 

;s  " 

Compensation 

0.541 

5  mm.                      °-37i 

37^  mm. 
25 

Huygenian 

0.290 

12^      " 

0.850 

N.  A.  =  0.92       °;^°0 
0.173 

180      mm. 
45         " 
15         " 

10 

Compensation 

0.270 
2  mm                        al86 

37^  mm. 
25        " 

Huygenian 

0.147 

12^       '« 

0.450 
0.251 
N.  A.  =  1.25  1         0.125 

0.088 

j 

180      mm. 
45 
15 

10 

Compensation 

FUNCTION    OF    AN   OBJECTIVE 

§  53.  Put  a  50  mm.  objective  on  the  microscope  or  screw  off  the 
front  combination  of  a  16  mm.,(^-in.),  and  put  the  back  combination 
on  the  microscope  for  a  low  objective. 

Place  some  printed  letters  or  figures  under  the  microscope,  and 
light  well.  In  place  of  an  ocular  put  a  screen  of  ground  glass,  or  a 
piece  of  lens  paper,  over  the  upper  end  of  the  tube  of  the  microscope.* 

*Ground  glass  may  be  very  easily  prepared  by  placing  some  fine  emery  or 
carborundum  between  two  pieces  of  glass,  wetting  it  with  water  and  then  rubbing 
the  glasses  together  for  a  few  minutes.  If  the  glass  becomes  too  opaque,  it  may 
be  rendered  more  translucent  by  rubbing  some  oil  upon  it. 


30  MICROSCOPE  AND  ACCESSORIES  \_CH.  I 

Lower  the  tube  of  the  microscope  by  means  of  the  coarse  adjust- 
ment until  the  objective  is  within  2-3  cm.  of  the  object  on  the  stage. 
Look  at  the  screen  on  the  top  of  the  tube,  holding  the  head  about  as 
far  from  it  as  for  ordinary  reading,  and  slowly  elevate  the  tube  by  means 
of  the  coarse  adjustment  until  the  image  of  the  letter  appears  on  the 
screen. 

The  image  can  be  more  clearly  seen  if  the  object  is  in  a  strong 
light  and  the  screen  in  a  moderate  light,  i.  e. ,  if  the  top  of  the  micro- 
scope is  shaded. 

The  letters  will  appear  as  if  printed  on  the  ground  glass  or  paper, 
but  will  be  inverted  ( Fig.  21). 

If  the  objective  is  not  raised  sufficiently,  and  the  head  is  held  too 
near  the  microscope,  the  objective  will  act  as  a  simple  microscope.  If 
the  letters  are  erect,  and  appear  to  be  down  in  the  microscope  and  not 
on  the  screen,  hold  the  head  farther  from  it,  shade  the  screen,  and 
raise  the  tube  of  the  microscope  until  the  letters  do  appear  on  the 
ground  glass. 

To  demonstrate  that  the  object  must  be  outside  the  principal  focus 
with  the  compound  microscope,  remove  the  screen  and  turn  the  tube  of 
the  microscope  directly  toward  the  sun.  Move  the  tube  of  the  micro- 
scope with  the  coarse  adjustment  until  the  burning  or  focal  point  is 
found  (§  6).  Measure  the  distance  from  the  paper  object  on  the  stage 
to  the  objective,  and  it  will  represent  approximately  the  principal 
focal  distance  (Figs.  10,  n).  Replace  the  screen  over  the  top  of  the 
tube,  no  image  can  be  seen.  Slowly  raise  the  tube  of  the  microscope 
and  the  image  will  finally  appear.  If  the  distance  between  the  object 
and  the  objective  is  now  taken,  it  will  be  found  considerably  greater 
than  the  principal  focal  distance  (compare  §  n). 

§  54.  Aerial  Image. — After  seeing  the  real  image  on  the  ground- 
glass,  or  paper,  use  the  lens  paper  over  about  half  of  the  opening  of 
the  tube  of  the  microscope.  Hold  the  eye  about  250  mm.  from  the 
microscope  as  before  and  shade  the  top  of  the  tube  by  holding  the  hand 
between  it  and  the  light,  or  in  some  other  way.  The  real  image  can 
be  seen  in  part  as  if  on  the  paper  and  in  part  in  the  air.  Move  the 
paper  so  that  the  image  of  half  a  letter  will  be  on  the  paper  and  half 
in  the  air.  Another  striking  experiment  is  to  have  a  small  hole  in  the 
paper  placed  over  the  center  of  the  tube  opening,  then  if  a  printed  word 
extends  entirely  across  the  diameter  of  the  tube  its  central  part  may  be 
seen  in  the  air,  the  lateral  parts  on  the  paper.  The  advantage  of  the 
paper  over  part  of  the  opening  is  to  enable  one  to  accommodate  the 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  31 

eyes  for  the  right  distance.  If  the  paper  is  absent  the  eyes  adjust 
themselves  for  the  light  circle  at  the  back  of  the  objective,  and  the 
aerial  image  appears  low  in  the  tube.  Furthermore  it  is  more  difficult 
to  see  the  aerial  image  in  space  than  to  see  the  image  on  the  ground- 
glass  or  paper,  for  the  eye  must  be  held  in  the  right  position  to  receive 
the  rays  projected  from  the  real  image,  while  the  granular  surface  of 
the  glass  and  the  delicate  fibres  of  the  paper  reflect  the  rays  irregularly, 
so  that  the  image  may  be  seen  at  almost  any  angle,  as  if  the  letters 
were  actually  printed  on  the  paper  or  glass. 

§  55.  The  Function  of  an  Objective,  as  seen  from  these  experi- 
ments, is  to  form  an  enlarged,  inverted,  real  image  of  an  object,  this 
image  being  formed  on  the  opposite  side  of  the  objective  from  the 
object  (Fig.  21). 

FUNCTION   OF   AN    OCULAR 

§  56.  Using  the  same  objective  as  for  §  53,  get  as  clear  an  image 
of  the  letters  as  possible  on  the  lens  paper  screen.  Look  at  the  image 
with  a  simple  microscope  (Fig.  17  or  1 8)  as  if  the  image  were  an  object. 

Observe  that  the  image  seen  through  the  simple  microscope  is 
merely  an  enlargement  of  the  one  on  the  screen,  and  that  the  letters 
remain  inverted,  that  is  they  appear  as  with  the  naked  eye  (§  n). 
Remove  the  screen  and  observe  the  aerial  image  with  the  tripod. 

Put  a  50  mm.  (A,  No.  i  or  2  in.),  ocular  i.  e.,  an  ocular  of  low 
magnification)  in  position  (§  48).  Hold  the  eye  about  10  to  20  milli- 
meters from  the  eye-lens  and  look  into  the  microscope.  The  letters 
will  appear  as  when  the  simple  microscope  was  used  (see  above),  the 
image  will  become  more  distinct  by  slightly  raising  the  tube  of  the 
microscope  with  the  coarse  adjustment. 

§  57.  The  Function  of  the  Ocular,  as  seen  from  the  above,  is 
that  of  a  simple  microscope,  viz. :  It  magnifies  the  real  image  formed 
by  the  objective  as  if  that  image  were  an  object.  Compare  the  image 
formed  by  the  ocular  (Fig.  21),  and  that  formed  by  a  simple  microscope 

(Fig-  38). 

It  should  be  borne  in  mind,  however,  that  the  rays  from  an  object 
as  usually  examined  with  a  simple  microscope,  extend  from  the  object 
in  all  directions,  and  no  matter  at  what  angle  the  simple  microscope  is 
held,  provided  it  is  sufficiently  near  and  points  toward  the  object,  an 
image  may  be  seen.  The  rays  from  a  real  image,  however,  are  continued 
in  certain  definite  lines  and  not  in  all  directions ;  hence,  in  order 
to  see  this  aerial  image  with  an  ocular  or  simple  microscope,  or 


MICROSCOPE  AND  ACCESSORIES 


\CH.  I 


in  order  to  see  the  aerial  image  with  the  unaided  eye,  the  simple  micro- 
scope, ocular  or  eye  must  be  in  the  path  of  the  rays  (Fig.  21.) 

FIG.  38.  Diagram  of  the  simple  microscope 
showing  the  course  of  the  rays  and  all  the  images, 
and  that  the  eye  forms  an  integral  part  of  it. 

A1  B*.  The  object  within  the  principal  focus. 
A*  B*.  The  virtual  image  on  the  same  side  of 
the  lens  as  the  object.  It  is  indicated  by  dotted 
lines,  as  it  has  no  actual  existence. 

B2  A2.  Retinal  image  of  the  object  (A1  S1}. 
The  virtual  image  is  simply  a  projection  of  the 
retinal  image  into  the  field  of  vision. 

Axis.  The  principal  optic  axis  of  the  micro- 
scope and  of  the  eye.  Cr.  Cornea  of  the  eye.  L. 
Crystalline  lens  of  the  eye.  R.  Ideal  refracting 
surface  at  which  all  the  refractions  of  the  eye  may 
be  assumed  to  take  place.  .5*.  .- ,  .^  3 

§  58.  The  field-lens  of  a  Huygenian  ocular  makes  the  real 
image  smaller  and  consequently  increases  the  size  of  the  field  ;  it  also 
makes  the  image  brighter  by  contracting  the  area  of  the  real  image. 
(Fig.  30.)  Demonstrate  this  by  screwing  off  the  field-lens  and  using 
the  eye-lens  alone  as  an  ocular,  refocusing  if  necessary.  Note  also  that 
the  image  is  bordered  by  a  colored  haze  (§7). 

When  looking  into  the  ocular  with  the  field-lens  removed,  the  eye 
should  not  be  held  so  close  to  the  ocular,  as  the  eye-point  is  consider- 
ably farther  away  than  when  the  field-lens  is  in  place. 

§  59.  The  eye-point. — This  is  the  point  above  the  ocular  or 
simple  microscope  where  the  greatest  number  of  emerging  rays  cross. 
Seen  in  profile,  it  may  be  likened  to  the  narrowest  part  of  an  hour 
glass.  Seen  in  section  (Fig.  30),  it  is  the  smallest  and  brightest 
light  circle  above  the  ocular.  This  is  called  the  eye-point,  for  if  the 
pupil  of  the  eye  is  placed  at  this  level,  it  will  receive  the  greatest 
number  of  rays  from  the  microscope,  and  consequently  see  the  largest 
field. 

Demonstrate  the  eye-point  by  having  in  position  an  objective  and 
ocular  as  above  (§  53).  Light  the  object  brightly,  focus  the  micro- 
scope, shade  the  ocular,  then  hold  some  ground-glass  or  a  piece  of  the 
lens  paper  above  the  ocular  and  slowly  raise  and  lower  it  until  the 
smallest  circle  of  light  is  found.  By  using  different  oculars  it  will  be 


CH.  /.]  MICROSCOPE  AND  ACCESSORIES  33 

seen  that  the  eye-point  is  nearer  the  eye-lens  in  high  than  in  low  ocu- 
lars, that  is  the  eye-point  is  nearer  the  eye-lens  for  an  ocular  of  small 
equivalent  focus  than  for  one  of  greater  focal  length. 

REFERENCES     FOR    CHAPTER    I 

In  chapter  X  will  be  given  a  bibliography,  with  full  titles,  of  the  works  and 
periodicals  referred  to. 

For  the  subjects  considered  in  this  chapter,  general  works  on  the  microscope 
may  be  consulted  with  great  advantage  for  different  or  more  exhaustive  treatment. 
The  most  satisfactory  work  in  English  is  Carpenter-Dallinger,  8th  Ed.  For  the 
history  of  the  microscope,  MayaH's  Cantor  Lectures  on  the  microscope  are  very 
satisfactory.  For  a  continuation  of  the  history  begun  by  Mayall  in  the  Cantor 
Lectures  see  Nelson,  Journal  of  the  Queckett  Micr.  Club,  and  the  Jour.  Roy. 
Micr.  Soc.,  1897-1901  —  .  Carpenter-Dallinger,  8th  Ed.  Petri,  Das  Mikroskop. 

The  following  special  articles  in  periodicals  may  be  examined  with  advantage  : 

Apochromatic  Objectives,  etc.  Dippel  in  Zeit.  wiss.  Mikr.,  1886,  p.  303  ;  also 
in  the  Jour.  Roy.  Micr.  Soc.,  1886,  pp.  316,  849,  mo,  ;  same,  1890,  p.  480  ;  Zeit.  f.. 
Instrumentenk. ,  1890,  pp.  1-6  ;  Micr.  Built.,  1891,  pp.  6-7. 

Tube-length,  etc.  Gage,  Proc.  Amer.  Soc.  Micrs.,  1887,  pp.  168-172  ;  also  in 
the  Microscope,  the  Jour.  Roy.  Micr.  Soc.,  and  in  Zeit,  wiss.  Mikr.,  1887-8. 
Bausch,  Proc.  Amer.  Soc.  Micrs.,  1890,  pp.  43-49  ;  also  in  the  Microscope,  1890, 
pp.  289-296. 

Aperture.  J.  D.  Cox,  Presidential  Address,  Proc.  Amer.  Soc.  Micrs.,  1884,  pp, 
5-39,  Jour.  Roy.  Micr.  Soc.,  iSSi,  pp.  303,  348,  365,  388  ;  1882,  pp.  300,  460  ;  1883, 
p.  790 ;  1884,  p.  20.  Czapski,  Theorie  der  optischen  Instrumente  nach  Abbe. 
See  also  references  in  \  34. 


The  Barnes  Dissecting  Microscope  (  The  Bausch  &  Lomb  Optical  Company). 


CHAPTER   II 


LIGHTING    AND    FOCUSING;     MANIPULATION    OF    DRY, 

ADJUSTABLE  AND  IMMERSION  OBJECTIVES  ;  CARE 

OF  THE  MICROSCOPE  AND  OF  THE  EYES  ; 

LABORATORY  MICROSCOPES 


APPARATUS   AND   MATERIAL   FOR    THIS    CHAPTER 

Microscope  supplied  with  plane  and  concave  mirror,  Achromatic  and  Abbe 
condensers,  dry,  adjustable  and  immersion  objectives,  oculars,  triple  nose-piece. 
Microscope  lamp  and  movable  condenser  (bull's  eye  or  other  form,  Fig.  53), 
Homogeneous  immersion  liquid  ;  Benzin,  alcohol,  distilled  water  ;  Mounted  prep- 
aration of  fly's  wing($  70);  Mounted  preparation  of  Pleurosigma  ($  77,  78).  Stage 
or  ocular  micrometer  (\  92);  Glass  slides  and  cover-glasses  (Ch.  VII);  10  per  ct. 
solution  of  salicylic  acid  in  95  per  ct.  alcohol  (\  92);  Preparation  of  stained  bac- 
teria ($  108);  Vial  of  equal  parts  olive  or  cotton  seed  oil  or  liquid  vaselin  and 
benzin  (\  112):  Double  eye  shade  (Fig.  60);  Screen  for  whole  microscope  (Fig.  59). 

FOCUSING 

$  60.  Focusing  is  mutually  arranging  an  object  and  the  microscope  so  that  a 
clear  image  may  be  seen. 

With  a  simple  , microscope  (In)  either  the  object  or  the  microscope  or  both 
may  be  moved  in  order  to  see  the  image  clearly,  but  with  the  compound  microscope 
the  object  more  conveniently  remains  stationary  on  the  stage,  and  the  tube  or 
body  of  the  microscope  is  raised  or  lowered  (frontispiece). 

In  general,  the  higher  the  power  of  the  whole  microscope  whether  simple  or 
compound,  the  nearer  together  must  the  object  and  objective  be  brought.  With 
the  compound  microscope,  the  higher  the  objective,  and  the  longer  the  tube  of 
the  microscope,  the  nearer  together  must  the  object  and  the  objective  be  brought. 
If  the  oculars  are  not  par-focal,  the  higher  the  magnification  of  the  ocular,  the 
nearer  must  the  object  and  objective  be  brought. 

$  61.  Working  Distance. — By  this  is  meant  the  space  between  the  simple  mi- 
croscope and  the  object,  or  between  the  front  lens  of  the  compound  microscope  and 
the  object,  when  the  microscope  is  in  focus.  This  working  distance  is  alwa3?s  con- 
siderably less  than  the  equivalent  focal  length  of  the  objective.  For  example, 
the  front-lens  of  a  6  mm.  or  %ih  in.  objective  would  not  be  6  millimeters  or  j^th 
inch  from  the  object  when  the  microscope  is -in  focus,  but  considerably  less  than 
that  distance.  If  there  were  no  other  reason  than  the  limited  working  distance  of 
high  objectives,  it  would  be  necessary  to  use  a  very  thin  cover-glass  over  the 


CH.  77] 


LIGHTING  AND  FOCUSING 


35 


object.  (See  \  24,  29. )  If  too  thick  covers  are  used  it  may  be  impossible  to  get 
an  objective  near  enough  an  object  to  get  it  in  focus.  For  objects  that  admit  of 
examination  with  high  powers  it  is  always  better  to  use  thin  covers. 

LIGHTING    WITH    DAYLIGHT 

\  62.  Unmodified  sunlight  should  not  be  employed  except  in  special  cases. 
North  light  is  best  and  most  uniform.  When  the  sky  is  covered  with  white  clouds 
the  light  is  most  favorable.  To  avoid  the  shadows  produced  by  the  hands  in 
manipulating  the  mirror,  etc. ,  it  is  better  to  face  the  light  ;  but  to  protect  the 
eyes  and  to  shade  the  stage  of  the  microscope  some  kind  of  screen  should  be 
used.  The  one  figured  in  (Fig.  62)  is  cheap  and  efficient.  If  one  dislikes  to  face 
the  window  or  lamp  it  is  better  to  sit  so  that  the  light  will  come  from  the  left  as  in 
reading. 

It  is  of  the  greatest  importance  and  advantage  for  one  who  is  to  use  the 
microscope  for  serious  work  that  he  should  comprehend  and  appreciate  thoroughly 
the  various  methods  of  illumination,  and  the  special  appearances  due  to  different 
kinds  of  illumination. 

Depending  on -whether  the  light  illuminating  an  object  traverses  the  object  or 
is  reflected  upon  it,  and  also  whether  the  object  is  symmetricallj*  lighted,  or 
lighted  more  on  one  side  than  the  other,  light  used  in  microscopy  is  designated  as 
reflected  and  transmitted,  axial  and  oblique. 


39 
FIGS.  39-40.     For  full  explanation  see  Figs.  22  and  23. 

\  63.  Reflected,  Incident  or  Direct  Light. — By  this  is  meant  light  reflected 
upon  the  object  in  some  way  and  then  irregularly  reflected  from  the  object  to  the 
microscope.  By  this  kind  of  light  objects  are  ordinarily  seen  by  the  unaided  eye, 
and  the  objects  are  mostly  opaque.  In  Vertebrate  Histology,  reflected  light  is  but 


36  LIGHTING  AND  FOCUSING  \_CH.  II 

little  used  ;  but  in  the  study  of  opaque  objects,  like  whole  insects,  etc.,  it  is  used 
a  great  deal.  For  low  powers,  ordinary  daylight  that  naturally  falls  upon  the 
object,  OF  is  reflected  or  condensed  upon  it  with  a  mirror  or  condensing  lens, 
answers  very  well.  For  high  powers  and  for  special  purposes,  special  illuminating 
apparatus  has  been  devised  (g  28).  (See  also  Carpenter-Dallinger,  Ch.  IV). 

§  64.  Transmitted  Light. — By  this  is  meant  light  which  passes  through  an 
object  from  the  opposite  side.  The  details  of  a  photographic  negative  are  in 
many  cases  only  seen  or  best  seen  by  transmitted  light,  while  the  print  made  from 
it  is  best  seen  by  reflected  light. 

Almost  all  objects  studied  in  Vertebrate  Histology  are  lighted  by  transmitted 
light,  and  they  are  in  some  way  rendered  transparent  or  semi-transparent.  The 
light  traversing  and  serving  to  illuminate  the  object  in  working  with  a  compound 
microscope  is  usually  reflected  from  a  plane  or  concave  mirror,  or  from  a  mirror  to 
a  condenser  (|  88),  and  thence  transmitted  to  the  object  from  below  (Figs.  48-51). 

\  65.  Axial  or  Central  Light. — By  this  is  understood  light  reaching  the  object, 
the  rays  of  light  being  parallel  to  each  other  and  to  the  optic  axis  of  the  micro- 
scope, or  a  diverging  or  converging  cone  of  light  whose  axial  ray  is  coincident  with 
the  optic  axis  of  the  microscope.  In  either  case  the  object  is  symmetrically 
illuminated. 

\  66.  Oblique  Light. — This  is  light  in  which  parallel  rays  from  a  plane  mirror 
form  an  angle  with  the  optic  axis  of  the  microscope  (Fig.  40).  Or  if  a  concave 
mirror  or  a  condenser  is  used,  the  light  is  oblique  when  the  axial  ray  of  the  cone 
of  light  forms  an  angle  with  the  optic  axis  (Fig.  40). 

DIAPHRAGMS 

$  67.  Diaphragms  and  their  Proper  Employment. — Diaphragms  are  opaque 
disks  with  openings  of  various  sizes,  which  are  placed  between  the  source  of  light 
or  mirror  and  the  object.  In  some  cases  an  iris  diaphragm  is  used,  and  then  the 
same  one  is  capable  of  giving  a  large  range  of  openings.  The  object  of  a  dia- 
phragm in  general,  is  to  cut  off  all  adventitious  light  and  thus  enable  one  to  light 
the  object  in  such  a  way  that  the  light  finally  reaching  the  microscope  shall  all 
come  from  the  object  or  its  immediate  vicinity.  The  diaphragms  of  a  condenser 
serve  to  vary  its  aperture  to  the  needs  of  each  object  and  each  objective. 

$  68.  Size  and  Position  of  Diaphragm  Opening. — When  no  condenser  is  used 
the  size  of  the  opening  in  the  diaphragm  should  be  about  that  of  the  front  lens 
of  the  objective.  For  some  objects  and  some  objectives  this  rule  may  be  quite 
widely  departed  from  ;  one  must  learn  by  trial. 

When  lighting  with  a  mirror  the  diaphragm  should  be  as  close  as  possible  to 
the  object  in  order,  (a)  that  it  may  exclude  all  adventitious  light  from  the  object  ; 
(b )  that  it  may  not  interfere  with  the  most  efficient  illumination  from  the  mirror 
by  cutting  off  a  part  of  the  illuminating  pencil.  If  the  diaphragm  is  a  considera- 
ble distance  below  the  object,  ( i )  it  allows  considerable  adventitious  light  to  reach 
the  object  and  thus  injures  the  distinctness  of  the  microscope  image  ;  (2)  it  pre- 
vents the  use  of  very  oblique  light  unless  it  swings  with  the  mirror  ;  ( 3 )  it  cuts  off 
a  part  of  the  illuminating  cone  from  a  concave  mirror.  On  the  other  hand,  even 
with  a  small  diaphragm,  the  whole  field  will  be  lighted. 


CH.  //]  LIGHTING  AND  FOCUSING  37 

With  an  illuminator  or  condenser  (Figs.  41,  48) ,  the  diaphragm  serves  to  narrow 
the  pencil  to  be  transmitted  through  the  condenser,  and  thus  to  limit  the  aperture 
(see  \  84).  Furthermore,  by  making  the  diaphragm  opening  eccentric,  oblique 
light  may  be  used,  or  by  using  a  diaphragm  with  a  slit  around  the  edge  ( central 
stop  diaphragm),  the  center  remaining  opaque,  the  object  may  be  lighted  with  a 
hollow  cone  of  light,  all  of  the  rays  having  great  obliquity.  In  this  way  the  so- 
called  dark -ground  illumination  may  be  produced  (|  92  ;  Fig.  51). 

ARTIFICIAL   ILLUMINATION 

|  69.  For  evening  work  and  for  certain  special  purposes,  artificial  illumina- 
tion is  employed.  A  good  petroleum  (kerosene)  lamp  with  flat  wick  has  been 
found  very  satisfactory,  also  an  incandescent  electric  or  Welsbach  light,  but  for 
brilliancy  and  for  the  actinic  power  necessary  for  very  rapid  photo-micrography 
(see  Ch.  VIII)  the  electric  arc  lamp  or  an  acetylene  lamp  serves  well.  Whatever 
source  of  artificial  light  is  employed,  the  light  should  be  brilliant  and  steady. 

LIGHTING    AND    FOCUSING  I    EXPERIMENTS 

§  70.  Lighting  with  a  Mirror. — As  the  following  experiments 
are  for  mirror  lighting  only,  remove  the  substage  condenser  if  present 
(see  §  79,  for  condenser).  Place  a  mounted  fly's  wing  under  the 
microscope,  put  the  16  mm.(^j  in.)  or  other  low  objective  in  position, 
also  a  low  ocular.  With  the  coarse  adjustment  lower  the  tube  of  the 
microscope  to  within  about  i  cm.  of  the  object.  Use  an  opening  in 
the  diaphragm  about  as  large  as  the  front  lens  of  the  objective  ; 
then  with  the  plane  mirror  try  to  reflect  light  up  through  the  diaphragm 
upon  the  object.  One  can  tell  when  the  field  (§  50)  is  illuminated,  by 
looking  at  the  object  on  the  stage,  but  more  satisfactorily  by  looking 
into  the  microscope.  It  sometimes  requires  considerable  manipulation 
to  light  the  field  well.  After  using  the  plane  side  of  the  mirror  turn 
the  concave  side  into  position  and  light  the  field  with  it.  As  the  con- 
cave mirror  condenses  the  light,  the  field  will  look  brighter  with  it  than 
with  the  plane  mirror.  It  is  especially  desirable  to  remember  that  the 
excellence  of  lighting  depends  in  part  on  the  position  of  the  diaphragm 
(§  68).  If  the  greatest  illumination  is  to  be  obtained  from  the  concave 
mirror,  its  position  must  be  such  that  its  focus  will  be  at  the  level  of 
the  object.  This  distance  can  be  very  easily  determined  by  finding  the 
focal  point  of  the  mirror  in  full  sunlight. 

§  71.  Use  of  the  Plane  and  of  the  Concave  Mirror. — The  mir- 
ror should  be  freely  movable,  and  have  a  plane  and  a  concave  face.  The 
concave  face  is  used  when  a  large  amount  of  light  is  needed,  the  plane 
face  wrhen  a  moderate  amount  is  needed  or  when  it  is  necessary  to  have 
parallel  rays  or  to  know  the  direction  of  the  rays. 


38  LIGHTING  AND  FOCUSING  [CH.  II 

§  72.  Focusing  with  Low  Objectives. — Place  a  mounted  fly's 
wing  under  the  microscope  ;  put  the  16  mm.  (^i  in.)  objective  in 
position,  and  also  the  lowest  ocular.  Select  the  proper  opening  in  the 
diaphragm  and  light  the  object  well  with  transmitted  light  (§  64,  68). 

Hold  the  head  at  about  the  level  of  the  stage,  look  toward  the 
window,  and  between  the  object  and  the  front  of  the  objective  ;  with 
the  coarse  adjustment  lower  the  tube  until  the  objective  is  within 
about  half  a  centimeter  of  the  object.  Then  look  into  the  microscope 
and  slowly  elevate  the  tube  with  the  coarse  adjustment.  The  image 
will  appear  dimly  at  first,  but  will  become  very  distinct  by  raising  the 
tube  still  higher.  If  the  tube  is  raised  too  high  the  image  will  become 
indistinct,  and  finally  disappear.  It  will  again  appear  if  the  tube  is 
lowered  the  proper  distance. 

When  the  microscope  is  well  focused  try  both  the  concave  and  the 
plane  mirrors  in  various  positions  and  note  the  effect.  Put  a  high 
ocular  in  place  of  the  low  one  (§43).  If  the  oculars  are  not  par- 
focal  it  will  be  necessary  to  lower  the  tube  somewhat  to  get  the  micro- 
scope in  focus.* 

Pull  out  the  draw-tube  4-6  cm.,  thus  lengthening  the  body  of  the 
microscope  ;  it  will  be  found  necessary  to  lower  the  tube  of  the  micro- 
scope somewhat.  (For  reason,  see  Fig.  58.) 

§  73.  Pushing  in  the  Draw-Tube.— To  push  in  the  draw-tube, 
grasp  the  large  milled  ring  of  the  ocular  with  one  hand,  and  the 
milled  head  of  the  coarse  adjustment  with  the  other,  and  gradually 
push  the  draw-tube  into  the  tube.  If  this  were  done  without  these 
precautions  the  objective  might  be  forced  against  the  object  and  the 
ocular  thrown  out  by  the  compressed  air. 

§  74.  Focusing  with  High  Objectives. — Employ  the  same 
object  as  before,  elevate  the  tube  of  the  microscope  and,  if  no  revolving 
nose-piece  is  present,  remove  the  16  mm.  (^  in.)  objective  as  indi- 
cated. Put  the  3  mm.(^  in.)  or  a  higher  objective  in  place,  and  use 
a  low  ocular. 


*Par-focal  oculars  are  so  constructed,  or  so  mounted,  that  those  of  different 
powers  may  be  interchanged  without  the  microscopic  image  becoming  wholly  out 
of  focus  (Fig.  31).  When  high  objectives  are  used,  while  the  image  may  be 
seen  after  changing  oculars,  the  instrument  nearly  always  needs  slight  focusing. 
With  low  powers  this  may  not  be  necessary. 

Objectives  are  also  now  commonly  mounted  in  the  triple  or  double  revolving 
nose-pieces  (.Figs.  36,  36  a)  so  that  if  one  of  the  objectives  is  in  focus  either  of  the 
others  will  be  approximately  in  focus  when  turned  into  position.  This  is  a  very 
great  convenience. 


CH.  //]  LIGHTING  AND  FOCUSING  39 

Light  well,  and  employ  the  proper  opening  in  the  diaphragm,  etc. 
(§  68).  Look  between  the  front  of  the  objective  and  the  object  as 
before  (§72),  and  lower  the  tube  with  the  coarse  adjustment  till  the 
objective  almost  touches  the  cover-glass  over  the  object.  Look  into 
the  microscope,  and  with  the  coarse  adjustment,  raise  the  tube  very 
slowly  until  the  image  begins  to  appear,  then  turn  the  milled  head  of 
the  fine  adjustment  (frontispiece),  first  one  way  and  then  the  other,  if 
necessary,  until  the  image  is  sharply  defined. 

In  practice  it  is  found  of  great  advantage  to  move  the  preparation 
slightly  while  focusing.  This  enables  one  to  determine  the  approach 
to  the  focal  point  either  from  the  shadow  or  the  color,  if  the  object  is  col- 
ored. With  high  powers  and  scattered  objects  there  might  be  no  object 
in  the  small  field  (see  §  50,  Fig.  37  for  size  of  field).  By  moving  the 
preparation  an  object  will  be  moved  across  the  field  and  its  shadow 
gives  one  the  hint  that  the  objective  is  approaching  the  focal  point.  It 
is  sometimes  desirable  to  focus  on  the  edge  of  the  cement  ring  or  on 
the  little  ring  made  by  the  marker  (see  Figs.  61-66). 

Note  that  this  high  objective  must  be  brought  nearer  the  object 
than  the  low  one,  and  that  by  changing  to  a  higher  ocular  (if  the  ocu- 
lars are  not  par-focal)  or  lengthening  the  tube  of  the  microscope  it 
will  be  found  necessary  to  bring  the  objective  still  nearer  the  object,  as 
with  the  low  objective.  (For  reason  see  Fig.  58.) 

§  75.  Always  Focus  Up,  as  directed  above.  If  one  lowers  the 
tube  only  when  looking  at  the  end  of  the  objective  as  directed  above, 
there  will  be  no  danger  of  bringing  the  objective  in  contact  with  the 
object,  as  may  be  done  if  one  looks  into  the  microscope  and  focuses 
down. 

When  the  instrument  is  well  focused,  move  the  object  around  in 
order  to  bring  different  parts  into  the  field.  It  may  be  necessary  to 
re-focus  with  the  fine  adjustment  every  time  a  different  part  is  brought 
into  the  field.  In  practical  work  one  hand  is  kept  on  the  fine  adjust- 
ment constantly,  and  the  focus  is  continually  varied. 

§  76.  Determination  of  Working  Distance. — As  stated  in  §  61, 
this  is  the  distance  between  the  front  lens  of  the  objective  and  the 
object  when  the  objective  is  in  focus.  It  is  always  less  than  the  equiv- 
lent  focal  length  of  the  objective. 

Make  a  wooden  wedge  10  cm.  long  which  shall  be  exceedingly  thin 
at  one  end  and  about  20  mm.  thick  at  the  other.  Place  a  slide  on  the 
stage  and  some  dust  on  the  slide.  Do  not  use  a  cover-glass.  Focus  the 
dust  carefully  first  with  the  low  then  with  the  high  objective.  When 


40  LIGHTING  AND  FOCUSING  [CH.  II 

the  objective  is  in  focus  push  the  wedge  under  the  objective  on  the 
slide  until  it  touches  the  objective.  Mark  the  place  of  contact  with  a 
pencil  and  then  measure  the  thickness  of  the  wedge  with  a  rule 
opposite  the  point  of  contact.  This  thickness  will  represent  very 
closely  the  working  distance.  For  measuring  the  thickness  of  the 
wedge  at  the  point  of  contact  for  the  high  objective  use  a  steel  scale 
ruled  in  ^ths  mm.  and  the  tripod  to  see  the  divisions.  Or  one  may 
use  a  cover-glass  measure  (Ch.  VIII)  for  determining  the  thickness  of 
the  wedge. 

For  the  higher  powers  if  one  has  a  microscope  in  which  the  fine  ad- 
justment is  graduated,  the  working  distance  may  be  readily  determined 
when  the  thickness  of  the  cover-glass  over  the  specimen  is  known,  as 
follows  :  Get  the  object  in  focus,  lower  the  tube  of  the  microscope,  un- 
til the  front  of  the  objective  just  touches  the  cover-glass.  Note  the 
position  of  the  micrometer  screw  and  slowly  focus  up  with  the  fine 
adjustment  until  the  object  is  in  focus.  The  distance  the  objective  was 
raised  plus  the  thickness  of  the  cover-glass  represents  the  working  dis- 
tance. For  example,  a  3  mm.  objective  after  being  brought  in  contact 
with  the  cover- glass  was  raised  by  the  fine  adjustment  a  distance  repre- 
sented by  1 6  of  the  divisions  on  the  head  of  the  micrometer  screw. 
Each  division  represented  .01  mm.,  consequently  the  objective  was 
raised  .  16  mm.  As  the  cover-glass  on  the  specimen  used  was  .  15  mm. 
the  total  working  distance  is  .16  +.15  =-31  mm. 

|  76a.  Free  Working  Distance. — In  the  microscope  catalog  of  Zeiss  there  is 
given  a  table  of  the  size  of  the  field  and  also  of  the  "free  working-distance."  This 
free  working-distance  is  the  space  between  the  lower  end  of  the  objective  and  the 
cover  glass  of  Ty^mm.  thickness,  when  the  objective  is  in  focus  on  an  object  imme- 
diately under  the  cover.  This  is  exceedingly  practical  information  for  a  possessor 
of  a  microscope,  and  it  is  hoped  that  the  other  opticians  will  adopt  the  suggestion. 
Naturally,  however,  the  free  working-distance  for  each  optician  should  be  reckoned 
from  the  top  of  the  cover  for  which  his  unadjustable  objectives  are  corrected.  If, 
for  example,  the  thickness  of  cover  for  which  an  objective  is  corrected  is  -^  mm. 
then  the  free  working-distance  should  be  that  between  the  top  of  this  and  the 
objective  when  the  objective  is  in  focus  on  an  object  under  the  cover.  (See  the 
table  of  cover-glass  thickness,  p.  14). 

CENTRAL   AND    OBLIQUE   LIGHT   WITH    A   MIRROR 

§  77.  Axial  or  Central  Light  (§  65).— Remove  the  condenser 
or  any  diaphragm  from  the  substage,  then  place  a  preparation  contain- 
ing minute  air  bubbles  under  the  microscope.  The  preparation  may 
be  easily  made  by  beating  a  drop  of  mucilage  on  a  slide  and  covering 


CH.  //]  LIGHTING  AND  FOCUSING  41 

it  (see  Ch.  III).  Use  a  3  mm.,(^  in.)  or  No.  7  objective  and  a  medium 
ocular.  Focus  the  microscope  and  select  a  very  small  bubble,  one 
whose  image  appears  about  i  mm.  in  diameter,  then  arrange  the  plane 
mirror  so  that  the  light  spot  in  the  bubble  appears  exactly  in  the 
center.  Without  changing  the  position  of  the  mirror  in  the  least, 
replace  the  air  bubble  preparation  by  one  of  Pleiirosigma  angulatum  or 
some  other  finely  marked  diatom.  Study  the  appearance  very  carefully. 

§  78.  Oblique  Light  (§  66). — Swing  the  mirror  far  to  one  side 
so  that  the  rays  reaching  the  object  may  be  very  oblique  to  the  optic 
axis  of  the  microscope.  Study  carefully  the  appearance  of  the  diatom 
with  the  oblique  light.  Compare  the  appearance  with  that  where  central 
light  is  used.  The  effect  of  oblique  light  is  not  so  striking  with  histo- 
logical  preparations  as  with  diatoms. 

It  should  be  especially  noted  in  §§  77,  78,  that  one  cannot  deter- 
mine the  exact  direction  of  the  rays  by  the  position  of  the  mirror. 
This  is  especially  true  for  axial  light  (  §77).  To  be  certain  the  light 
is  axial  some  such  test  as  that  given  in  §  77  should  be  applied.  (See 
also  Ch.  Ill,  under  Air-bubbles.) 

CONDENSERS  OR  ILLUMINATORS* 

§  79.  These  are  lenses  or  lens-systems  for  the  purpose  of  illuminat- 
ing with  transmitted  light  the  object  to  be  studied  with  the  microscope. 

For  the  highest  kind  of  investigation  their  value  cannot  be  over- 
estimated. They  may  be  used  either  with  natural  or  artificial  light, 
and  should  be  of  sufficient  numerical  aperture  to  satisfy  objectives  of 
the  widest  angle. 


*No  one  has  stated  more  cleaily,  or  appreciated  more  truly  the  value  of  cor- 
rect illumination  and  the  methods  of  obtaining  it  than  Sir  David  Brewster,  1820, 
1831.  He  says  of  illumination  in  general  :  "The  art  of  illuminating  microscopic 
objects  is  not  of  less  importance  than  that  of  preparing  them  for  observation. " 
"The  eye  should  be  protected  from  all  extraneous  light,  and  should  not  receive  any 
of  the  light  which  proceeds  from  the  illuminating  center,  excepting  that  portion 
of  it  which  is  transmitted  through  or  reflected  from  the  object."  So  likewise  the 
value  and  character  of  the  substage  condenser  was  thoroughly  understood  and 
pointed  out  by  him  as  follows  :  "I  have  no  hesitation  in  saying  that  the  apparatus 
for  illumination  requires  to  be  as  perfect  as  the  apparatus  for  vision,  and  on  this 
account  I  would  recommend  that  the  illuminating  lens  should  be  perfectly  free  of 
chromatic  and  spherical  aberration,  and  the  greatest  care  be  taken  to  exclude  all 
extraneous  light  both  from  the  object  and  from  the  eye  of  the  observer."  See  Sir 
David  Brewster's  treatise  on  the  Microscope,  1837,  pp.  136,  138,  146,  and  the 
Edinburgh  Journal  of  Science,  new  series,  No.  n  (1831)  p.  83. 


42  LIGHTING  AND  POCUSING  {_CH.  II 

It  is  of  the  greatest  advantage  to  have  the  sub-stage  condenser 
mounted  so  that  it  may  be  easily  moved  up  or  down  under  the  stage. 
The  iris  diaphragm  is  so  convenient  that  it  should  be  furnished  in  all 
cases,  and  there  should  be  marks  indicating  the  N.  A.  of  the  condenser 
utilized  with  different  openings.  Finally  the  condenser  should  be 
supplied  with  central  stops  for  dark-ground  illumination  (§  92)  and 
with  blue  and  neutral  tint  glasses  to  soften  the  glare  when  artificial 
light  is  used  (§  89,  93). 

Condensers  or  Illuminators  fall  into  two  great  groups,  the 
Achromatic,  giving  a  large  aplanatic  cone,  and  Non-achromatic, 
giving  much  light,  but  a  relatively  small  aplanatic  cone  of  light. 

§  80.  Achromatic -Condenser. — It  is  still  believed  by  all  expert 
microscopists  that  the  contention  of  Brewster  was  right,  and  the  con- 
denser to  give  the  greatest  aid  in  elucidating  microscopic  structure 
must  approach  in  excellence  the  best  objectives.  That  is,  it  should  be 
as  free  as  possible  from  spherical  and  chromatic  aberration,  and  there- 
fore would  transmit  to  the  object  a  very  large  aplanatic  cone  of  light. 
Such  condensers  are  especially  recommended  for  photo-micrography  by 
all,  and  those  who  believe  in  getting  the  best  possible  image  in  every 
case  are  equally  strenuous  that  achromatic  condensers  should  be  used 
for  all  work.  Unfortunately  good  condensers  like  good  objectives  are 
expensive,  and  student  microscopes  as  well  as  many  others  are  usually 
supplied  with  the  non-achromatic  condensers  or  with  none. 

Many  excellent  achromatic  condensers  have  been  made,  but  the 
most  perfect  of  all  seems  to  be  the  apochromatic  of  Powell  and  Lealand 
(Carpenter-Dallinger,  p.  302).  To  attain  the  best  that  was  possible 
many  workers  have  adopted  the  plan  of  using  objectives  as  condensers. 
A  special  substage  fitting  is  provided  with  the  proper  screw  and  the 
objective  is  put  into  position,  the  front  lens  being  next  the  object.  As 
will  be  seen  below  (§  83-84),  the  full  aperture  of  an  objective  can 
rarely  be  used,  and  for  histological  preparations  perhaps  never,  so  that 
an  objective  of  greater  equivalent  focus,  i.  <?. ,  lower  power  is  used  for 
the  condenser  than  the  one  on  the  microscope.  It  is  much  more  con- 
venient, however,  to  have  a  special  condenser  with  iris  diaphragm  or 
special  diaphragms  so  that  one  may  use  any  aperture  at  will,  and  thus 
satisfy  the  conditions  necessary  for  lighting  different  objects  for  the 
same  objective  and  for  lighting  with  objectives  of  different  apertures. 
An  excellent  condenser  of  this  form  has  been  produced  by  Zeiss  (Fig. 
41).  It  has  a  total  numerical  aperture  of  i.oo,  and  an  aplanatic  aper- 
ture of  0.65. 


CH.  //]  LIGHTING  AND  FOCUSING  43 

FIG.  41.  Zeiss'  Achromatic  Conden- 
ser, c.  s.  c.  s.  Centering  screws  for 
changing  the  position  of  the  condenser 
and  making  its  axis  continuous  with 
that  of  the  microscope.  A  segment  of 
the  condenser  is  cut  away  to  show  the 
combinations  of  lenses.  For  very  low 
powers  the  upper  lens  is  sometimes 
screwed  off.  There  is  an  iris  dia- 
phragm between  the  middle  and  lower 
combinations.  ( Zeiss '  Catalog ) . 

§  81.  Centering  the  Condenser. — To  get  the  best  possible 
illumination  for  bringing  out  in  the  clearest  manner  the  minute  details 
of  a  microscopic  object  two  conditions  are  necessary,  viz.:  The  princi- 
pal optic  axis  of  the  condenser  must  be  continuous  with  that  of  the 
microscope  (see  frontispiece)  and  the  object  must  be  in  the  focus  of  the 
condenser,  i  e. ,  at  the  apex  of  the  cone  of  light  given  by  the  condenser. 

The  centering  is  most  conveniently  accomplished  as  follows 
although  daylight  may  be  used  with  almost  equal  facility.  A  very 
small  diaphragm  is  put  below  the  condenser.  (If  the  Zeiss  achromatic 
condenser  is  used,  the  diaphragm  of  the  Abbe  illuminator  serves  for 
this.  If  there  is  no  pin-hole  diaphragm  one  can  be  made  of  stiff, 
black  paper.  Care  must  be  taken,  however,  to  make  the  opening  ex- 
actly central.  This  is  best  accomplished  by  putting  the  paper  disc  over 
the  iris  or  metal  diaphragms  and  then  making  the  hole  in  the  center  of 
the  small  circle  uncovered  by  the  metal  diaphragm.  For  the  hole  a  fine 
needle  is  best).  Light  well  and  lower  the  objective  so  that  it  is  at 
about  its  working-distance  from  the  top  of  the  condenser.  If  now  the 
condenser  is  lowered  or  racked  away  from  the  objective  the  image  of 
the  diaphragm  will  appear.  If  the  opening  is  not  central  it  should  be 
made  so  by  using  the  centering  screws  of  the  condenser. 

A  better  plan  than  to  lower  the  condenser  to  focus  the  image  of  the 
diaphragm,  is  to  raise  the  body  of  the  microscope  slowly  with  the  coarse 
adjustment.  It  is  almost  impossible  to  make  apparatus  so  accurate  that 
two  parts  like  the  body  of  the  microscope  and  the  substage,  each  work- 
ing on  different  sliding  surfaces,  shall  continue  in  exactly  the  same 
plane.  So  one  will  find  that  if  the  condenser  be  accurately  centered 
with  the  condenser  lowered,  and  then  the  condenser  be  racked  up  close 
to  "the  stage  and  the  image  of  the  diaphragm  opening  brought  again 
into  focus  by  racking  up  the  body  of  the  microscope,  it  will  not  be 


44 


LIGHTING  AND  FOCUSING 


\_CH. 


accurately  centered  in  most  cases.  For  this  reason  it  is  advised  that 
the  condenser  be  left  in  position  close  to  the  stage  and  the  tube  of  the 
microscope  be  used  to  focus  the  diaphragm  exactly  as  in  ordinary 
work. 

FIG.  42 .  Shows  that  the  optic  axis  of 
the  condenser  does  not  coincide  with  that 
of  the  microscope.  ( D) .  Image  of  the 
diaphragm  of  the  condenser  shown  at 
one  side  of  the  field  of  view. 

FIG.  43.  Shows  the  image  of  the 
diaphragm  (D]  in  the  center  of  the  field 
of  the  microscope,  and  thus  the  coin- 
cidence of  the  axis  of  the  condenser  with 
that  of  the  microscope. 


.Exc 


FIG.  42 


FIG.  43 


§  82.  Centering  the  Image  of  the  Source  of  Illumination.— 
For  the  best  results  it  is  not  only  necessary  that  the  condenser  be  pro- 
perly centered,  but  that  the  object  to  be  studied  should  be  in  the  image 
of  the  source  of  illumination  and  that  this  should  also  be  centered 
(Figs.  44,  45).  After  the  condenser  itself  is  centered  the  iris  diaphragm 
is  opened  to  its  full  extent  or  the  diaphragm  carrier  turned  wholly 
aside.  A  transparent  specimen  like  the  fly's  wing  is  put  under  the 
microscope  and  focused.  The  condenser  is  then  turned  up  and  down 
until  the  image  of  the  flame  is  apparently  on  the  specimen.  If  this 
cannot  be  accomplished  the  relative  position  of  the  lamp  and  condenser 
is  not  correct  and  should  be  so  changed  that  the  image  of  the  edge  of 
the  flame  is  sharply  defined.  This  image  must  also  be  centered.  This 
is  easily  accomplished  by  manipulation  of  the  mirror  or,  if  a  lamp  is 
used,  by  changing  the  position  of  the  lamp  or  of  the  bull's  eye 

(Fig-  53). 

§83.     Proper    Numerical    Aperture  of  the    Condenser. — As 

stated  above,  the  aperture  of  the  condenser  should  have  a  range  by 
means  of  properly  selected  diaphragms  to  meet  the  requirements  of  all 
objectives  from  the  lowest  to  those  of  the  highest  aperture.  It  is 
found  in  practice  that  for  diatoms,  etc.,  the  best  images  are  obtained 
when  the  object  is  lighted  with  a  cone  which  fills  about  three- fourths 
of  the  diameter  of  the  back  lens  of  the  objective  with  light  but  for 
histological  and  other  preparations  of  lower  refractive  power  only  one- 
half  or  one-third  the  aperture  often  gives  the  most  satisfactory  images 
(§  34). 


CH.  77] 


LIGHTING  AND  FOCUSING 


45 


FIG.  44.  Shows  the  image  of  the 
flame  (Ft.)  in  the  center  (€}  of  the 
field  of  the  microscope  and  illuminat- 
ing the  object. 

FIG.  45.  Shows  the  image  of  the 
flame  (Fl.}  at  one  side  of  the  centre 
(Exc.}  and  not  properly  illuminating 
the  object. 


Exc 


FIG.  44. 


FIG.  45.  * 

To  determine  this  in  any  case  focus  upon  some  very  transparent 
object,  take  out  the  ocular,  look  down  the  tube  at  the  back  lens.  If  less 
than  three-fourths  of  the  back  lens  is  lighted,  increase  the  opening  in 
the  diaphragm— if  more  than  three-fourths  diminish  it.  For  some 
objects  it  is  advantageous  to  use  less  than  three-fourths  of  the  aper- 
ture. Experience  will  teach  the  best  lighting  for  special  cases. 


Obj 


O 


Obj 


Til 


Ilium 


FIG.  46. 


FIG.  47. 


FIGS.  46-47.     Figures  showing  the  dependence  of  the  objective  upon  the  ilium 
inating  cone  of  the  condenser  (Nelson}. 

FIG.  46  (A).     The  illuminating  cone  from  the  condenser  {I Hum}.     This  is 
seen  to  be  just  sufficient  to  fill  the  objective  (Obj}. 

(B}.     The  back  lens  of  the  objective  entirely  filled  with  light,  showing  that  the 
numerical  aperture  of'  the  illuminator  is  equal  to  that  of  the  objective. 

FIG.  47  (A}.  In  this  figure  the  illuminating  conefrome  the  cotidenser  (Ilium.} 
is  seen  to  be  insufficient  to  fill  the  objective  (Obj}. 

(B}.     The  back   lens  of  the  objective  only  partly  filled  with  light,  due  to  the 
restricted  aperture  of  the  illuminator. 

§  84.  Aperture  of  the  Illuminating  Cone  and  the  Field.— It 
is  to  be  remarked  that  with  a  very  small  source  of  light  the  entire  aper- 
ture of  the  objective  may  be  filled  if  a  proper  illuminator  or  condenser 
is  used.  The  aperture  depends  on  the  diaphragm  used  with  the  con- 
denser. And  the  size  of  the  diaphragm  must  be  directly  as  the  aper- 
ture of  the  objective.  That  is,  it  is  just  the  reverse  of  the  rule  for 
diaphragms  where  no  condenser  is  used  (§  67) ;  for  there  the  diaphragm 


46  LIGHTING  AND  FOCUSING  [CH.  II 

is  made  large  for  low  powers,  and  consequently  low  apertures,  while 
with  the  condenser  the  diaphragm  is  made  small  for  low  and  large  for 
high  powers  as  the  aperture  is  greater  in  the  high  powers  of  a  given 
series  of  objectives.  It  is  very  instructive  to  demonstrate  this  by  using 
a  1 6  mm.  objective  and  opening  the  diaphragm  of  the  condenser  till  the 
back  lens  is  just  filled  with  light.  Then  if  one  uses  a  3  or  4  mm.  ob- 
jective it  will  be  seen  that  the  back  lens  of  the  higher  objective  is  only 
partly  filled  with  light  and  to  fill  it  the  diaphragm  must  be  much  more 
widely  opened. 

With  a  condenser,  then,  the  diaphragm  has  simply  to  regulate  the 
aperture  of  the  illuminating  cone,  and  has  nothing  to  do  with  lighting 
a  large  or  a  small  field. 

With  the  condenser  there  are  two  conditions  that  must  be  fulfilled, 
— the  proper  aperture  must  be  used,  and  that  is  determined  by  the  dia- 
phragm, and  secondly  the  whole  field  must  be  lighted.  The  latter  is 
accomplished  by  using  a  larger  source  of  light,  as  the  face  instead  of 
the  edge  of  a  lamp  flame,  or  by  lowering  or  raising  the  condenser  so 
that  the  object  is  not  in  the  focus  of  the  condenser,  but  above  or  below 
it,  and  therefore  lighted  by  a  converging  or  diverging  beam  where  the 
Tight  is  spread  over  a  greater  area  (Figs.  48-51,  §  88). 

§  85.  Non- Achromatic  Condenser. — Of  the  non-achromatic 
condensers  or  illuminators,  the  Abbe  condenser  or  illuminator  is  the 
one  most  generally  used.  From  its  cheapness  it  is  also  much  more  com- 
monly used  than  the  achromatic  condenser.  It  consists  of  two  or  three 
very  large  lenses  and  transmits  a  cone  of  light  of  1.20  N.  A.  to  1.40  N. 
A.,  but  the  aberrations,  both  spherical  and  chromatic,  are  very  great  in 
both  forms.  Indeed,  so  great  are  they  that  in  the  best  form  of  three 
lenses  with  an  illuminating  cone  of  1.40  N.  A.,  the  aplanatic  cone 
transmitted  is  only  0.5,  and  it  is  the  aplanatic  cone^  which  is  of  real  use 
in  microscopic  illumination  where  details  are  to  be  studied.  There  is 
no  doubt,  however,  that  the  results  obtained  with  a  non-achromatic 
condenser  like  the  Abbe  are  much  more  satisfactory  than  with  no  con- 
denser. The  highest  results  cannot  be  attained  with  it,  however. 
(Carpenter-Dallinger,  p.  309). 

§  86.  Arrangement  of  the  Condenser. — The  proper  position  of 
the  illuminator  for  high  objectives  is  one  in  which  the  beam  of  light 
traversing  it  is  brought  to  a  focus  on  the  object.  If  parallel  rays  are 
reflected  from  the  plane  mirror  to  it,  they  will  be  focused  only  a  few 
millimeters  above  the  upper  lens  of  the  condenser  ;  consequently  the 
illuminator  should  be  about  on  the  level  of  the  top  of  the  stage  and 


CH.  77]  LIGHTING  AND  FOCUSING  47 

therefore  almost  in  contact  with  the  lower  surface  of  the  slide.  For 
some  purposes  when  it  is  desirable  to  avoid  the  loss  of  light  by  reflec- 
tion or  refraction,  a  drop  of  water  or  homogeneous  immersion  fluid  is 
put  between  the  slide  and  condenser,  forming  the  so-called  immersion 
illuminator.  This  is  necessary  only  with  objectives  of  high  power 
and  large  aperture  or  for  dark-ground  illumination. 

§87.  Centering  the  Condenser. — The  illuminator  should  be 
centered  to  the  optic  axis  of  the  microscope,  that  is  the  optic  axis  of 
the  condenser  and  of  the  microscope  should  coincide.  Unfortunately 
there  is  extreme  difficulty  in  determining  when  the  Abbe  illuminator  is 
centered.  Centering  is  approximated  as  follows  :  Put  a  pin-hole  dia- 
phragm— that  is  a  diaphragm  with  a  small  central  hole — over  the  end 
of  the  condenser  (Fig.  52),  the  central  opening  should  appear  to  be  in 
the  middle  of  the  field  of  the  microscope.  If  it  does  not  the  condenser 
should  be  moved  from  side  to  side  by  loosening  the  centering  screws 
until  it  is  in  the  center  of  the  field.  In  case  no  pin-hole  diaphragm 
accompanies  the  condenser,  one  may  put  a  very  small  drop  of  ink,  as 
from  a  pen-point,  on  the  center  of  the  upper  lens  and  look  at  it  with 
the  microscope  to  see  if  it  is  in  the  center  of  the  field.  If  it  is  not, 
the  condenser  should  be  adjusted  until  it  is.  When  the  condenser  is 
centered  as  nearly  as  possible  remove  the  pin-hole  diaphragm  or  the 
spot  of  ink.  The  microscope  and  illuminator  axes  may  not  be  entirely 
coincident  even  when  the  center  of  the  upper  lens  appears  in  the  cen- 
ter of  the  field,  as  there  may  be  some  lateral  tilting  of  the  condenser,  but 
the  above  is  the  best  the  ordinary  worker  can  do,  and  unless  the 
mechanical  arrangements  of  the  illuminator  are  very  deficient,  it  will 
be  very  nearly  centered. 

It  is  to  be  hoped  that  the  opticians  will  devise  some  kind  of 
mounting  for  this  the  most  cemmonly  used  condenser  whereby  it  may 
be  centered  as  described  for  the  achromatic  condenser  instead  of  by  the 
crude  methods  described  above.  If  the  condenser  mounting  regularly 
possessed  centering  screws  as  in  the  microscope  of  Watson  &  Sons  and 
there  were  a  centering  diaphragm  in  the  proper  position  so  that  its  im- 
age could  be  projected  into  the  field  of  view,  the  operation  would  be 
very  simple.  If,  further,  the  condensers  of  Powell  and  Lealand  were 
selected  as  models  the  condensers  need  not  be  so  bulky,  and  would  still 
retain  all  their  efficiency. 

Fortunately  the  Royal  Microscopical  Society  of  London  which  has 
done  so  much  toward  standardizing  microscopical  apparatus  has  recently 
proposed  as  a  standard  size  for  the  substage  fitting  for  the  condenser  of 
1.527  in.=  38.786  mm.  (see  §  46). 


4^  LIGHTING  AND  FOCUSING  \CH.  II 

§  88.  Mirror  and  Light  for  the  Abbe  Condenser. — It  is  best  to 
use  light  with  parallel  rays.  The  rays  of  daylight  are  practically  par- 
allel ;  it  is  best  therefore  to  employ  the  plane  mirror  for  all  but  the 
lowest  powers.  If  low  powers  are  used  the  whole  field  might  not  be 
illuminated  with  the  plane  mirror  when  the  condenser  is  close  to  the 
object  ;  furthermore,  the  image  of  the  window  frame,  objects  outside 
the  building,  as  trees,  etc. ,  would  appear  with  unpleasant  distinctness 
in  the  field  of  the  microscope.  To  overcome  these  defects  one  can 
lower  the  condenser  and  thus  light  the  object  with  a  diverging  cone  of 
light,  or  use  the  concave  mirror  and  attain  the  same  end  when  the  con- 
denser is  close  to  the  object  (Fig.  48). 

§  89.  Artificial  Light. — If  one  uses  lamp  light,  it  is  recommend- 
ed that  a  large  bull's  eye  be  placed  in  such  a  position  between  the 
light  and  the  mirror  that  parallel  rays  fall  upon  the  mirror  or  in  some 
cases  an  image  of  the  lamp  flame.  If  one  does  not  have  a  bull's  eye 
the  concave  mirror  may  be  used  to  render  the  rays  less  divergent.  It 
may  be  necessary  to  lower  the  illuminator  somewhat  in  order  to  illum- 
inate the  object  in  its  focus. 

ABBE   CONDENSER  :    EXPERIMENTS 

§  90.  Abbe  Condenser,  Axial  and  Oblique  Light. — Use  a  dia- 
phragm a  little  larger  than  the  front  lens  of  the  3  mm.  (^  in.)  objec- 
tive, have  the  illuminator  on  the  level,  or  nearly  on  the  level  of  the 
upper  surface  of  the  stage,  and  use  the  plane  mirror.  Be  sure  that 
the  diaphragm  carrier  is  in  the  notch  indicating  that  it  is  central  in 
position.  Use  the  Pleurosigma  as  object.  Study  carefully  the  appear- 
ance of  the  diatom  with  this  central  light,  then  make  the  diaphragm 
eccentric  so  as  to  light  with  oblique  light  (§78).  The  differences  in 
appearance  will  probably  be  even  more  striking  than  with  the  mirror 
alone. 

§  91.  Lateral  Swaying  of  the  Image. — Frequently  in  study- 
ing an  object,  especially  with  a  high  power,  it  will  appear  to  sway 
from  side  to  side  in  focusing  up  or  down.  A  glass  stage  micrometer  or 
fly's  wing  is  an  excellent  object.  Make  the  light  central  or  axial  and 
focus  up  and  down  and  notice  that  the  lines  simply  disappear  or  grow 
dim.  Now  make  the  light  oblique,  either  by  making  the  diaphragm 
opening  eccentric  or  if  simply  a  mirror  is  used,  by  swinging  the  mirror 
sidewise.  On  focusing  up  and  down,  the  lines  will  sway  from  side  to 
side.  What  is  the  direction  of  apparent  movement  in  focusing  down 


CH.  //] 


LIGHTING  AND  FOCUSING 


49 


with  reference  to  the  illuminating  ray  ?  What  in  focusing  up  ?  If  one 
understands  the  experiment  it  may  sometimes  save  a  great  deal  of  con- 
fusion. (See  under  testing  the  microscope  for  swaying  with  central 
light  §  119.) 

§  92.  Dark-Ground  Illumination. — When  an  object  is  lighted 
with  rays  of  a  greater  obliquity  than  can  get  into  the  front  lens  of  the 
objective,  the  field  will  appear  dark  (Fig.  51).  If  now  the  object  is 


FIGS.  48-51.  Sectional  views  of  the  Abbe  Illuminator  of  1.20  N.  A.  showing 
various  methods  of  illumination  (\  oo)  Fig.  48,  axial  light  with  parallel  rays, 
Fig.  49,  oblique  light.  Fig.  50,  axial  light  with  converging  beam.  Fig.  51,  dark- 
ground  illumination  with  a  central  stop  diaphragm. 

Axis.  The  optic  axis  of  the  illuminator  and  of  the  microscope.  The  illumi- 
nator is  centered,  that  is  its  optic  axis  is  a  prolongation  of  the  optic  axis  of  the 
microscope. 

S.  Axis.  Secondary  axis.  In  oblique  light  the  central  ray  passes  along  a 
secondary  axis  of  the  illuminator,  and  is  therefore  oblique  to  the  principal  axis. 

D.  D.  Diaphragms.  These  are  placed  in  sectional  and  in  face  views.  The 
diaphragm  is  placed  between  the  mirror  and  the  illuminator.  In  Fig.  49  the  open- 
ing is  eccentric  for  oblique  light,  and  in  Fig.  51  the  opening  is  a  narrow  ring,  the 
central  part  being  stopped  out,  thus  giving  rise  to  dark-ground  illumination  ($  92). 

Obj.  Obj.     The  front  of  the  objective. 

composed  of  fine  particles,  or  is  semi-transparent,  it  will  refract  or 
reflect  the  light  which  meets  it,  in  such  a  way  that  a  part  of  the  very 


50  LIGHTING  AND  FOCUSING  [CH.  II 

oblique  rays  will  pass  into  the  objective,  hence  as  light  reaches  the 
objective  only  from  the  object,  all  the  surrounding  field  will  be  dark 
and  the  object  will  appear  like  a  self-luminous  one  on  a  dark  back- 

FIG.  52.     An  Abbe  Condenser  in  its  mounting 
{The  Bausch  &  Lomb  Optical  Company}. 

ground.  This  form  of  illumination  is  most 
successful  with  low  powers.  It  is  well  to 
make  the  illuminator  immersion  for  this 
experiment,  (see  §  105). 

(A)  With  the  Mirror— Remove  all  the 
diaphragms  so  that  very  oblique  light  may 
be  used,  employ  a  stage  micrometer  in 
which  the  lines  have  been  filled  with  graph- 
ite, use  a  1 6  mm.  (^  in.)  objective,  and  when  the  light  is  sufficiently 
oblique  the  lines  will  appear  something  like  streaks  of  silver  on  a 
black  back-ground.  A  specimen  like  that  described  below  in  (B)  may 
also  be  used. 

(B)  With  the  Abbe  Condenser. — Have  the  illuminator  so  that  the 
light  is  focused  on  the  object  (see  §  86)  and  use  a  diaphragm  with 
the  annular  opening  (Fig.  51);  employ  the  same  objective  as  in 
(A).  For  object  place  a  drop  of  10  %  solution  of  salicylic  acid  in  95  % 
alcohol  on  the  middle  of  a  slide  ;  it  will  crystallize.  The  crystals  will 
appear  brilliantly  lighted  on  a  dark  back-ground.  Put  in  an  ordinary 
diaphragm  and  make  the  light  oblique  by  making  the  diaphragm 
eccentric.  The  same  specimen  may  also  be  tried  with  a  mirror  and 
oblique  light.  In  order  to  appreciate  the  difference  between  this  dark- 
ground  and  ordinary  transmitted-light  illumination,  use  an  ordinary 
diaphragm  and  observe  the  crystals. 

A  very  striking  and  instructive  experiment  may  be  made  by  add- 
ing a  very  small  drop  of  the  solution  to  the  dried  preparation,  putting 
it  under  the  microscope  quickly,  lighting  for  dark-ground  illumination 
and  then  watching  the  crystallization. 

ARTIFICIAL   ILLUMINATION 

§  93.  For  evening  work  and  for  regions  where  daylight  is  not 
sufficiently  brilliant,  artificial  illumination  must  be  employed.  Fur- 
thermore, for  the  most  critical  investigation  of  bodies  with  fine  mark- 
ings like  diatoms,  artificial  light  has'been  found  superior  to  daylight. 

A  petroleum  (kerosene)  lamp  with  flat  wick  gives  a  satisfactory 
light.  It  is  recommended  that  instead  of  the  ordinary  glass  chimney, 


CH.  //] 


LIGHTING  AND  FOCUSING 


one  made  of  metal  with  a  slit- opening  covered  with  an  oblong  cover- 
glass  is  more  satisfactory,  as  the  source  of  light  is  more  restricted. 
Very  excellent  results  may  be  obtained,  however,  with  the  ordinary 
bed-room  lamp  furnished  with  the  usual  glass  chimney. 

The  new  acetylene  light  promises  to  be  excellent  for  micro- 
scopic observation  and  for  photo-micrography.  (See  under  photo- 
micrography.) 


FIG.  53.     i. 
separate  stand. 


Lamp   with  slit-opening  in  metal  chimney. 
3.  Screen  showing  image  of  flame. 


2.     BuWs  eye  on 


Whenever  possible  the  edge  of  the  flame  is  turned  toward  the 
microscope,  the  advantage  of  this  arrangement  is  the  great  brilliancy, 
due  to  the  greater  thickness  of  the  flame  in  this  direction. 

§  94.  Mutual  Arrangement  of  Lamp,  Bull's  Eye  and  Micro- 
scope.— To  fulfill  the  conditions  given  above,  namely,  that  the  object 
be  illuminated  by  the  image  of  the  source  of  illumination  the  lamp 
must  be  in  such  a  position  that  the  condenser  projects  a  sharp  image 
of  the  flame  upon  the  object  (Fig.  53),  and  only  by  trial  can  this  posi- 
tion be  determined.  In  some  cases  it  is  found  advantageous  to  discard 
the  mirror  and  allow  the  light  from  the  bull's  eye  to  pass  directly  into 
the  condenser.  This  method  is  especially  excellent  in  photomicro- 
graphy (see  Ch.  VIII). 

§  95.  Illuminating  the  Entire  Field. — With  low  objectives 
and  large  objects,  the  entire  object  might  not  be  illuminated  if  the 
above  method  were  strictly  followed  ;  in  this  case  turn  the  lamp  so 
that  the  flame  is  oblique,  or  if  that  is  not  sufficient,  continue  to  turn 
the  lamp  until  the  full  width  of  the  flame  is  used.  If  necessary  the 


LIGHTING  AND  FOCUSING 


\CH.  II 


condenser  may   be    lowered,    and    the    concave    mirror    used.     (See 
also  §84.) 

REFRACTION   AND    COLOR   IMAGES 

\  96.  Refraction  Images  are  those  mostly  seen  in  studying  microscopic 
objects.  They  are  the  appearances  produced  by  the  refraction  of  the  light  on 
entering  and  on  leaving  an  object.  They  therefore  depend  (a)  on  the  form  of  the 
object,  (b)  on  the  relative  refractive  powers  of  object  and  mounting  medium. 
With  such  images  the  diaphragm  should  not  be  too  large  (see  §83). 

If  the  color  and  refractive  index  of  the  object  were  exactly  like  the  mount- 
ing medium  it  could  not  be  seen.  In  most  cases  both  refractive  index  and  color 
differ  somewhat,  there  is  then  a  combination  of  color  and  refraction  images  which 
is  a  great  advantage.  This  combination  is  generally  taken  advantage  of  in  histol- 
ogy. The  air  bubble  in  $  77  is  an  example  of  a  purely  refractive  image. 


54 


N' 


FIGS.  54-56.  Diagrams  illustrating  refraction  in  different  media  and  at  plane 
and  curved  surfaces.  In  each  case  the  denser  medium  is  represented  by  line  shad- 
ing and  the  perpendicular  or  normal  to  the  refracting  surface  is  represented  by  the 
dotted  line  N-N' ,  the  refracted  ray  by  the  bent  line  A  C. 

\  97 .  Refraction. — Lying  at  the  basis  of  microscopical  optics  is  refraction ,  which 
is  illustrated  by  the  above  figures.  It  means  that  light  passing  from  one  medium 
to  another  is  bent  in  its  course.  Thus  in  Fig.  54  light  passing  from  air  into  water 
does  not  continue  in  a  straight  line  but  is  bent  toward  the  normal  N-NX,  the 
bending  taking  place  at  the  point  of  contact  of  the  air  and  water ;  that  is,  the  ray 
of  light  A  B  entering  the  water  at  B  is  bent  out  of  its  course,  extending  to  C 
instead  of  Cx. 

Conversely,  if  the  ray  of  light  is  passing  from  water  into  air,  on  reaching  the 
air  it  is  bent_/r0w  the  normal,  the  ray  C  B  passing  to  A  and  not  in  a  straight  line 
to  Cx/.  By  comparing  Figs.  55,  56  in  which  the  denser  medium  is  crown  glass  in- 
stead of  water,  the  bending  of  the  rays  is  seen  to  be  greater  as  crown  glass  is 
denser  than  water. 

It  has  been  found  by  physicists  that  there  is  a  constant  relation  between  the 
angle  taken  by  the  ray  in  the  rarer  medium  and  that  taken  by  the  ray  in  the 
denser  medium.  The  relationship  is  expressed  thus  :  Sine  of  the  angle  of  inci- 


CH.  77]  LIGHTING  AND  FOCUSING  53 

dence  divided  by  the  sine  of  the  angle  of  refraction  equals  the  index  of  refraction. 

In  the  figures,    \  °  .7  =  index  of    refraction.     Worked    out  completely   in 

oin  C  lj  J\ 


Fig.  54, 


the  index  of  refraction  from  air  to  water  is  1.33.  (See  $  33.)  In  Figs.  55-56, 
illustrating  refraction  in  crown  glass,  the  angles  being  given,  the  problem  is  easily 
solved  as  just  illustrated.  (For  table  of  natural  sines  see  third  page  of  cover  ;  for 
interpolation,  |  32). 

|  98.  Absolute  Index  of  Refraction.  —  This  is  the  index  of  refraction  obtained 
when  the  incident  ray  passes  from  a  vacuum  into  a  given  medium.  As  the  index 
of  the  vacuum  is  taken  as  unity,  the  absolute  index  of  any  substance  is  always 
greater  than  unity.  For  many  purposes,  as  for  the  object  of  this  book, 
air  is  treated  as  if  it  were  a  vacuum,  and  its  index  is  called  unity,  but  in  reality 
the  index  of  refraction  of  air  is  about  3  ten-thousandths  greater  than  unity. 
Whenever  the  refractive  index  of  a  substance  is  given,  the  absolute  index  is 
meant  unless  otherwise  stated.  For  example,  when  the  index  of  refraction  of 
water  is  said  to  be  1.33,  and  of  crown  glass  1.52,  etc.,  these  figures  represent  the 
absolute  index,  and  the  incident  ray  is  supposed  to  be  in  a  vacuum. 

\  99.  Relative  Index  of  Refraction.  —  This  is  the  index  of  refraction  between 
two  contiguous  media,  as  for  example  between  glass  and  diamond,  water  and 
glass,  etc.  It  is  obtained  by  dividing  the  absolute  index  of  refraction  of  the  sub- 
stance containing  the  refracted  ray,  by  the  absolute  index  of  the  substance  trans- 
mitting the  incident  ray.  For  example,  the  relative  index  from  water  to  glass  is 
1.52  divided  by  1.33.  If  the  light  passed  from  glass  to  water  it  would  be,  1.33 
divided  by  1.52. 

By  a  study  of  the  figures  showing  refraction,  it  will  be  seen  that  the  greater  the 
refraction  the  less  the  angle  and  consequently  the  less  the  sine  of  the  angle,  and  as 
the  refracti  on  between  two  media  is  the  ratio  of  the  sines  of  the  angles  of  incidence 

and  refraction  (          -  )  ,  it  will  be  seen  that  whenever  the  sine  of  the  angle  of  refrac- 
Vsin  rj\ 

tion  is  increased  by  being  in  a  less  refractive  medium,  the  index  of  refraction  will 
show  a  corresponding  decrease  and  vice  versa.  That  is  ihe  ratio  of  the  sines  of  the 
angles  of  incidence  and  refraction  of  any  two  contiguous  substances  is  inversely  as 
the  refractive  indices  of  those  substances.  The  formula  is  : 

/  Sine  of  angle  of  incident  ray  \       /  Index  of  refraction  of  refracting  medium  \ 
\  Sine  of  angle  of  refracted  ray  /       \  Index  of  refraction  of  incident  medium  / 

Abbreviated  (  -      -  |  =  |  r  -.  )  •  BV  means  of  this  general  formula  one  can 

\sm  rj      \index  ij 

solve  any  problem  in  refraction  whenever  three  factors  of  the  problem  are  known. 

The  universality  of  the  law  may  be  illustrated  by  the  following  cases  : 

(A)    Light  incident  in  a  vacuum  or  in  air,  and  entering  some  denser  medium, 

as  water,  glass,  diamond,  etc. 

/         Sine  of  angle  made  by  the  ray  in  air         \  _  /Index  of  ref.of  denser  med.  \ 
\  Sine  of  angle  made  by  ray  in  denser  medium  /       \     Index  of  ref.  of  air  (  i)     / 

If  the  dense  substance  were  glass  (   -      -  )  —  (  —  ^—  J  .     If  the  two  media  were 

\sin  r)~  V     i      / 


54  LIGHTING  AND  FOCUSING  [CH.  II 

water   and   glass,   the   incident    light   being    in   water  the   formula  would  be  : 

(  —. )  =  (  )  •     If  the  incident  ray  were  in  glass  and  the  refracted  ray  in 

\sm  r)      V  1.33  / 

water  :    (  — )  —  (  -£s-  }  -  And  similarly  for  any  two  media  ;  and   as   stated 

Vsin  r)      V  1.52  / 

above  if  any  three  of  the  factors  are  given  the  fourth  may  be  readily  found. 

§  ico.  Critical  Angle  and  Total  Reflection. — In  order  to  understand  the  Wol- 
laston  camera  lucida  (Ch.  IV)  and  other  totally  reflecting  apparatus,  it  is  necessary 
briefly  to  consider  the  critical  angle. 

The  critical  angle  is  the  greatest  angle  that  a  ray  of  light  in  the  denser  of  two 
contiguous  media  can  make  with  the  normal  and  still  emerge  into  the  less  refrac- 
tive medium.  On  emerging  it  will  form  an  angle  of  90°  with  the  normal,  and  if 
the  substances  are  liquids,  the  refracted  ray  will  be  parallel  with  the  surface  of  the 
denser  medium. 

Total  Reflection. — In  case  the  incident  ray  in  the  denser  medium  is  at  an  angle 
with  the  normal  greater  than  the  critical  angle^  it  will  be  totally  reflected  at  the 
surface  of  the  denser  medium,  that  surface  acting  as  a  perfect  mirror.  By  consult- 
ing the  figures  it  will  be  seen  that  there  is  no  such  thing  as  a  critical  angle  and 
total  reflection  in  the  rarer  of  two  contiguous  media. 

To  find  the  critical  angle  in  the  denser  of  two  contiguous  media  : — 

Make  the  angle  of  refraction  (z.  e. ,  the  angle  in  the  rarer  of  the  two  media) 

/  sin  i  \       /  index  r  \ 

90    and  solve  the  general  equation  :    (  —. I  =  (  -. — , r  I .     Let  the   two  sub- 

\sin  r/       \index  z  / 

stances  be  water  and  air,  then  the  sine  of  r  (90°)  is  i,  the  index  of  air  is  i,  that  of 
water  1.33,  whence     (-      -  1  =  1  -      -  j    or  sin  z' —  75 1 -j- .     This  is  the  sine  of 

48°  +  ,  and  whenever  the  ray  in  the  water  is  at  an  angle  of  more  than  48°  it  will 
not  emerge  into  the  air,  but  be  totally  reflected  back  into  the  water. 

The  case  of  a  ray  passing  from  crown  glass  into  the  water  : 

/  _         sin  i  \  _  /  index  water  (1.33)  \       /  sin  i  \       /  1.33  \ 

\sin  r  (sin  90°=  i)/       \index  glass  (i.  52)7  °r  \     i     /       \  1.527' 
whence  sin  z  =.875   sine  of  critical   angle   in   glass   covered    with    water.     The 
corresponding  angle  is  approximately  61°. 

|  101.  Color  Images. — These  are  images  of  objects  which  are  strongly  col- 
ored and  lighted  with  so  wide  an  aperture  that  the  refraction  images  are  drowned 
in  the  light.  Such  images  are  obtained  by  removing  the  diaphragm  or  by  using  a 
larger  opening.  This  method  of  illumination  is  especially  applicable  to  the  study 
of  deeply  stained  bacteria.  (See  below.  §  108). 

ADJUSTABLE   WATER   AND    HOMOGENEOUS   OBJECTIVES 
EXPERIMENTS 

§  102.  Adjustment  for  Objectives. — As  stated  above  ($  24),  the  aberration 
produced  by  the  cover-glass  (Fig.  57),  is  compensated  for  by  giving  the  combina- 
tions in  the  objective  a  different  relative  position  than  they  would  have  if  the 
objective  were  to  be  used  on  uncovered  objects.  Although  this  relative  position 
cannot  be  changed  in  unadjustable  objectives,  one  can  secure  the  best  results  .of 


CH.  //] 


LIGHTING  AND  FOCUSING 


55 


which  the  objective  is  capable  by  selecting  covers  of  the  thickness  for  which  the 
objective  was  corrected.  (See  table  p.  14.)  Adjustment  maybe  made  also  by 
increasing  the  tube-length  for  covers  thinner  than  the  standard  and  by  shortening 
the  tube-length  for  covers  thicker  than  the  standard  (Fig.  58). 

FIG.  57.  Effect  of  the  cover-glass 
on  the  rays  from  the  object  to  the 
objective  (Ross}. 

Axis.  The  projection  of  the  optic 
axis  of  the  microscope. 

F.  Focal  or  axial  point  of  the 
objective. 

F'  and  F" .  Points  on  the  axis 
where  rays  2  and  j  appear  to  originate 
if  traced  backward  after  emerging 
from  the  upper  side  of  the  cover-glass. 

In  learning  to  adjust  objectives,  it  is  best  for  the  student  to  choose  some 
object  whose  structure  is  well  agreed  upon  ,  and  then  to  practice  lighting  it,  shad- 
ing the  stage  and  adjusting  the  objective,  until  the  proper  appearance  is  obtained. 
The  adjustment  is  made  by  turning  a  ring  or  collar  which  acts  on  a  screw  and 
increases  or  diminishes  the  distance  between  the  systems  of  lenses,  usually  the 
front  and  the  back  systems  (Fig.  40). 

§  103.  General  Directions. — (A)  The  thinner  the  cover-glass,  the 
further  must  the  system  of  lenses  be  separated,  i.  e.,  the  ad  justing  collar 
is  turned  nearer  the  zero  or  the  mark  "uncovered,"  and  conversely;  (B) 
the  thicker  the  cover-glass  the  closer  together  are  the  systems  brought 
by  turning  the  adjusting  collar  from  the  zero  mark.  This  also  increases 
the  magnification  of  the  objective  (Ch.  IV). 

The  following  specific  directions  for  making  the  cover-glass  adjust- 
ment are  given  by  Mr.  Wenham  (Carpenter,  yth  Ed.,  p.  166).  "Select 
any  dark  speck  or  opaque  portion  of  the  object,  and  bring  the  outline 
into  perfect  focus  ;  then  lay  the  finger  on  the  milled-head  of  the  fine 
motion,  and  move  it  briskly  backwards  and  forwards  in  both  directions 
from  the  first  position.  Observe  the  expansion  of  the  dark  outline  of 
the  object,  both  when  within  and  when  without  the  focus.  If  the 
greater  expansion  or  coma  is  when  the  object  is  without  the  focus,  or 
farthest  from  the  objective  \i.  e.,  in  focusing  up],  the  lenses  must  be 
placed  further  asunder,  or  toward  the  mark  uncovered  [the  adjusting 
collar  is  turned  toward  the  zero  mark  as  the  cover-glass  is  too  thin  for 
the  present  adjustment].  If  the  greater  expansion  is  when  the  object 
is  within  the  focus,  or  nearest  the  objective  [z.  e.,  in  focusing  down], 
the  lenses  must  be  brought  closer  together,  or  toward  the  mark  covered 
[i.  e.,  the  adjusting  collar  should  be  turned  away  from  the  zero  mark, 


56  LIGHTING  AND  FOCUSING  {_CH.  II 

the  cover-glass  being  too  thick  for  the  present  adjustment] ."  In  most 
objectives  the  collar  is  gradiiated  arbitrarily,  the  zero  (O}  mark  represent- 
ing the  position  for  uncovered  objects.  Other  objectives  have  the  collar 
graduated  to  correspond  to  the  various  thicknesses  of  cover-glasses  for  which 
the  objective  may  be  adjtisted.  This  seems  to  be  an  admirable  plan  ;  then 
if  one  knows  the  thickness  of  the  cover-glass  on  the  preparation  (Ch.  VIII) 
the  adjusting  collar  may  be  set  at  a  corresponding  mark,  and  one  will  feel 
confident  that  the  adjustment  will  be  approximately  correct.  It  is  then 
only  necessary  for  the  observer  to  make  the  slight  adjustment  to  compensate 
for  the  mounting  medium  or  any  variation  from  the  standard  length  of  the 
tube  of  the  microscope.  In  adjusting  for  variations  of  the  length  of 
the  tube  from  the  standard  it  should  be  remembered  that  :  (A)  If  the 
tube  of  the  microscope  is  longer  than  the  standard  for  which  the  ob- 
jective was  corrected,  the  effect  is  approximately  the  same  as  thicken- 
ing the  cover-glass,  and  therefore  the  system  of  the  objective  must  be 
brought  closer  together,  i.  e.,  the  adjusting  collar  must  be  turned  away 
from  the  zero  mark.  (B)  If  the  tube  is  shorter  than  the  standard  for 
which  the  objective  is  corrected,  the  effect  is  approximately  the  same  as 
diminishing  the  thickness  of  the  cover-glass,  and  the  systems  must 
therefore  be  separated  (Fig.  40). 

In  using  the  tube-length  for  cover  correction  Shorten  the  tube  for 
too  thick  covers  and  Lengthen  the  tube  for  too  thin  covers. 

Furthermore,  whatever  the  interpretation  by  different  opticians  of 
what  should  be  included  in  "tube-length,"  and  the  exact  length  in  mil- 
limeters, its  importance  is  very  great  ;  for  each  objective  gives  the  most 
perfect  image  of  which  it  is  capable  with  the  "tube-length"  for  which 
it  is  corrected,  and  the  more  perfect  the  objective  the  greater  the  ill- 
effects  on  the  image  of  varying  the  "tube-length"  from  this  standard. 
The  plan  of  designating  exactly  what  is  meant  by  "tube-length,"  and 
engraving  on  each  objective  the  "tube-length"  for  which  it  is  corrected, 
is  to  be  commended,  for  it  is  manifestly  difficult  for  each  worker  with 
the  microscope  to  find  out  for  himself  for  what  "tube-length"  each  of 
his  objectives  was  corrected.  (See  Ch.  X). 

§  104.  Water  Immersion  Objectives. — Put  a  water  immersion 
objective  in  position  (§  47)  and  the  fly's  wing  for  object  under  the 
microscope.  Place  a  drop  of  distilled  water  on  the  cover-glass,  and 
with  the  coarse  adjustment  lower  the  tube  till  the  objective  dips  into 
the  water,  then  light  the  field  well  and  turn  the  fine  adjustment  one 
way  and  another  till  the  image  is  clear.  Water  immersions  are  exceed- 
ingly convenient  in  studying  the  circulation  of  the  blood,  and  for 


CH.  77] 


LIGHTING  AND  FOCUSFNG 


57 


many  other  purposes  where  aqueous  liquids  are  liable  to  get  on  the 
cover-glass.  If  the  objective  is  adjustable,  follow  the  directions  given 
in  §  103. 


FIG.  58.  Figure  to  show  that  in  lengthening  the  tube  of  the  microscope  the  ob- 
ject must  be  brought  nearer  the  principal  focus  or  center  of  the  lens.  It  will  be  seen 
by  consulting  the  figure  that  in  shortening  the  tube  of  the  microscope  the  object 
must  be  removed  farther  from  the  center  of  the  lens.  By  consulting  the  figure 
showing  the  effect  of  the  cover-glass  (Fig.  57)  it  will  be  seen  that  the  effect  of  the 
cover-glass  is  to  bring  the  object  nearer  the  objective,  and  the  thicker  the  cover  the 
nearer  is  the  object  brought  to  the  objective.  As  shortening  the  tube  serves  to  remove 
the  object,  it  neutralizes  the  effect  of  the  thick  cover,  and  if  the  cover  is  so  thin  that 
it  does  not  elevate  the  object  enough  for  the  corrections  of  the  objective,  then  an  in- 
crease in  the  tube-length  will  correct  the  defect. 


58  LIGHTING  AND  FOCUSING  \_CH.  II 

When  one  is  through  using  a  water  immersion  objective,  remove  it 
from  the  microscope  and  with  some  lens  paper  wipe  all  the  water  from 
the  front  lens.  Unless  this  is  done  dust  collects  and  sooner  or  later 
the  front  lens  will  be  clouded.  It  is  better  to  use  distilled  water  to 
avoid  the  gritty  substances  that  are  liable  to  be  present  in  natural 
waters,  as  these  gritty  particles  might  scratch  the  front  lens. 

HOMOGENEOUS   IMMERSION   OBJECTIVES  :    EXPERIMENTS 

§  105.  As  stated  above,  these  are  objectives  in  which  a  liquid  of 
the  same  refractive  index  as  the  front  lens  of  the  objective  is  placed 
between  the  front  lens  and  the  cover-glass. 

§  106.  Tester  for  Homogeneous  Liquid. — In  order  that  full 
advantage  be  derived  from  the  homogeneous  immersion  principle,  the 
liquid  employed  must  be  truly  homogeneous.  To  be  sure  that  such  is 
the  case,  one  may  use  a  tester  like  that  constructed  by  the  Gundlach 
Optical  Co. ,  then  if  the  liquid  is  too  dense  it  may  be  properly  diluted 
and  vice  versa.  For  the  cedar  oil  immersion  liquid,  the  density  may 
be  diminished  by  the  addition  of  pure  cedar  wood  oil.  The  density 
may  be  increased  by  allowing  it  to  thicken  by  evaporation.  (See  H. 
L.  Smith,  Proc.  Amer.  Soc.  Micr.,  1885,  p.  83,  and  Ch.  X). 

§  107.  Refraction  Images. — Put  a  2  mm.(T^-th  in.)  homogeneous 
immersion  objective  in  position,  employ  an  illuminator.  Use  some 
histological  specimen  like  a  muscular  fiber  as  object,  make  the  dia- 
phragm opening  about  3  mm.  in  diameter,  add  a  drop  of  the  homo- 
geneous immersion  liquid  and  focus  as  directed  in  §  74.  The  object 
will  be  clearly  seen  in  all  details  by  the  unequal  refraction  of  the  light 
traversing  it.  The  difference  in  color  between  it  and  the  surrounding 
medium  will  also  increase  the  sharpness  of  the  outline.  If  an  air  bub- 
ble preparation  (§  77)  were  used,  one  would  get  pure,  refraction 
images.  • 

§  108.  Color  Images. — Use  some  stained  bacteria  as  Bacillus 
tuberculosis  for  object.  Put  a  drop  of  the  immersion  liquid  on  the 
cover-glass  or  the  front  lens  of  the  homogeneous  objective.  Remove 
the  diaphragms  from  the  illuminator  or  in  case  the  iris  diaphragm  is 
used,  open  to  its  greatest  extent.  Focus  the  objective  down  so  that 
the  immersion  fluid  is  in  contact  with  both  the  front  lens  and  the  cover- 
glass,  then  with  the  fine  adjustment  get  the  bacteria  in  focus.  They 
will  stand  out  as  clearly  defined  colored  objects  on  a  bright  field. 


CH.  //]  LIGHTING  AND  FOCUSING  59 


jo    cm 


FIG.  59.  Screen  for  shading  the  microscope  and 
the  face  of  the  observer.  This  is  very  readily  con- 
structed as  shown  in  the  figure  by  supporting  a  wire 
in  a  disc  of  lead,  iron,  or  heavy  wood.  The  screen  is 
then  completed  by  hanging  over  the  bent  wire,  cloth  or 
manilla  paper  30x40  cm.  The  lower  edge  of  the 
screen  should  be  a  little  below  the  stage  of  the  micro- 
scope and  the  upper  edge  high  enough  to  screen  the 
eyes  of  the  observer. 

§  109.  Shading  the  Object.— To  get  the 
clearest  image  of  an  object  no  light  should 
reach  the  eye  except  from  the  object.  A  hand- 
kerchief or  a  dark  cloth  wound  around  the 

objective  will  serve  the  purpose.  Often  the  proper  effect  may  be  ob- 
tained by  simply  shading  the  top  of  the  stage  with  the  hand  or  with  a 
piece  of  bristol  board.  Unless  one  has  a  very  favorable  light  the  shading 
of  the  object  is  of  the  greatest  advantage,  especially  with  homogeneous 
immersion  objectives.  The  screen  (Fig.  59)  is  the  most  satisfactory 
means  for  this  purpose,  as  the  entire  microscope  above  the  illuminating 
apparatus  is  shaded. 

§  no.  Cleaning  Homogeneous  Objectives. — After  one  is 
through  with  a  homogeneous  objective,  it  should  be  carefully  cleaned  as 
follows  :  Wipe  off  the  homogeneous  liquid  with  a  piece  of  the  lens 
paper  (§  114),  then  if  the  fluid  is  cedar  oil,  wet  one  corner  of  a  fresh 
piece  in  benzin  or  chloroform  and  wipe  the  front  lens  with  it.  Imme- 
diately afterward  wipe  with  a  dry  part  of  the  paper.  The  cover-glass 
of  the  preparation  can  be  cleaned  in  the  same  way.  If  the  homogen- 
eous liquid  is  a  glycerin  mixture  proceed  as  above,  but  use  water  to 
remove  the  last  traces  of  glycerin. 

CARE   OF   THE    MICROSCOPE 

§111.  The  microscope  should  be  handled  carefully  and  kept  per- 
fectly clean.  The  oculars  and  objectives  should  never  be  allowed  to 
fall. 

When  not  in  use  keep  it  in  a  place  as  free  as  possible  from  dust. 

All  parts  of  the  microscope  should  be  kept  free  from  liquids, 
especially  from  acids,  alkalies,  alcohol,  benzin,  turpentine  and 
chloroform. 

§  112.  Care  of  the  Mechanical  Parts. — To  clean  the  mechan- 
ical parts  put  a  small  quantity  of  some  fine  oil  (olive  oil  or  liquid  vas- 


60  LIGHTING  AND  FOCUSING  \_Cff.  II 

elin  and  benzin,  equal  parts),  on  a  piece  of  chamois  leather  or  on  the 
lens  paper,  and  rub  the  parts  well,  then  with  a  clean  dry  piece  of  the 
chamois  or  paper  wipe  off  most  of  the  oil.  If  the  mechanical  parts 
are  kept  clean  in  this  way  a  lubricator  is  rarely  needed.  Where  op- 
posed brass  surfaces  "cut,"  i.  <?.,  when  from  the  introduction  of  some 
gritty  material,  minute  grooves  are  worn  in  the  opposing  surfaces,  giv- 
ing a  harsh  movement,  the  opposing  parts  should  be  separated,  care- 
fully cleaned  as  described  above  and  any  ridges  or  prominences  scraped 
down  with  a  knife.  Where  the  tendency  to  "cut"  is  marked,  a  very 
slight  application  of  equal  parts  of  beeswax  and  tallow,  well  melted 
together,  serves  a  good  purpose. 

In  cleaning  lacquered  parts,  benzin  alone  answers  well,  but  it 
should  be  quickly  wiped  off  with  a  clean  piece  of  the  lens  paper.  Do 
not  use  alcohol  as  it  dissolves  the  lacquer. 

§  113.  Care  of  the  Optical  Parts. — These  must  be  kept  scrupu- 
lously clean  in  order  that  the  best  results  may  be  obtained. 

Glass  surfaces  should  never  be  touched  with  the  fingers,  for  that 
will  soil  them. 

The  glass  of  which  the  lenses  are  made  is  quite  soft,  consequently 
it  is  necessary  that  only  soft,  clean  cloth  or  paper  be  used  in  wiping 
them. 

Whenever  an  objective  is  left  in  position  on  a  microscope,  or  when 
several  are  attached  by  means  of  a  revolving  nose-piece,  an  ocular 
should  be  left  in  the  upper  end  of  the  tube  to  prevent  dust  from  falling 
down  upon  the  back  lens  of  the  objective. 

§  114.  Lens  Paper. — The  so-called  Japanese  filter  paper,  which 
from  its  use  with  the  microscope,  I  have  designated  lens  paper,  has 
been  used  in  the  author's  laboratory  for  the  last  sixteen  years  for  clean- 
ing the  lenses  of  oculars  and  objectives,  and  especially  for  removing 
the  fluid  used  with  immersion  objectives.  Whenever  a  piece  is  used 
once  it  is  thrown  away.  It  has  proved  more  satisfactory  than  cloth  or 
chamois,  because  dust  or  sand  is  not  present  ;  and  from  its  bibulous 
character  it  is  very  efficient  in  removing  liquid  or  semi-liquid  substances. 

§  115.  Dust  may  be  removed  with  a  camel's  hair  brush,  or  by 
wiping  with  the  lens  paper. 

Cloudiness  may  be  removed  from  the  glass  surfaces  by  breathing 
on  them,  then  wiping  quickly  with  a  soft  cloth  or  the  lens  paper. 

Cloudiness  on  the  inner  surfaces  of  the  ocular  lenses  may  be  re- 
moved by  unscrewing  them  and  wiping  as  directed  above.  A  high 
objective  should  never  be  taken  apart  by  an  inexperienced  person. 


CH.  //]  LIGHTING  AND  FOCUSING  61 

If  the  cloudiness  cannot  be  removed  as  directed  above,  moisten  one 
corner  of  the  cloth  or  paper  with  95  per  cent,  alcohol,  wipe  the  glass  first 
with  this,  then  with  the  dry  cloth  or  the  paper. 

Water  may  be  removed  with  soft  cloth  or  the  paper. 

Glycerin  may  be  removed  with  cloth  or  paper  saturated  with  dis- 
tilled water  ;  remove  the  water  as  above. 

Blood  or  other  albuminous  material  may  be  removed  while  fresh 
with  a  moist  cloth  or  paper,  the  same  as  glycerin.  If  the  material 
has  dried  on  the  glass,  it  may  be  removed  more  readily  by  adding  a 
small  quantity  of  ammonia  to  the  water  in  which  the  cloth  is  moistened, 
(water  100  cc.,  ammonia  i  cc). 

Canada  Balsam,  damar,  paraffin,  or  any  oily  substance  may  be  re- 
moved with  a  cloth  or  paper  wet  with  chloroform,  benzin  or  xylene. 
The  application  of  these  liquids  and  their  removal  with  a  soft  dry  cloth 
or  paper  should  be  as  rapid  as  possible,  so  that  none  of  the  liquid  will 
have  time  to  soften  the  setting  of  the  lenses. 

Shellac  Cement  may  be  removed  by  the  paper  or  a  cloth  moistened 
in  95  per  cent,  alcohol. 

Brunswick  Black,  Gold  Size,  and  all  other  substances  soluble  in 
chloroform,  etc.,  maybe  removed  as  directed  for  balsam  and  damar. 

In  general,  use  a  solvent  of  the  substance  on  the  glass  and  wipe  it 
off  quickly  with  a  fresh  piece  of  the  lens  paper. 

It  frequently  happens  that  the  upper  surface  of  the  back  combina- 
tion of  the  objective  becomes  dusty.  This  may  be  removed  in  part  by 
a  brush,  but  more  satisfactorily  by  using  a  piece  of  the  soft  paper  loosely 
twisted.  When  most  of  the  dust  is  removed  some  of  the  paper  may  be 
put  over  the  end  of  a  pine  stick  (like  a  match  stick)  and  the  glass  sur- 
face carefully  wiped. 

CARE   OF   THE   EYES 

§  1 1 6.  Keep  both  eyes  open,  using  the  eye-screen  if  necessary  (Figs. 
60,  6oa);  and  divide  the  labor  between  the  two  eyes,  i.  e.,  use  one  eye 
for  observing  the  image  awhile  and  then  the  other.  In  the  begin- 
ning it  is  not  advisable  to  look  into  the  microscope  continuously  for 
more  than  half  an  hour  at  a  time.  One  never  should  work  with  the 
microscope  after  the  eyes  feel  fatigued.  After  one  becomes  accustomed 
to  microscopic  observation  he  can  work  for  several  hours  with  the 
microscope  without  fatiguing  the  eyes.  This  is  due  to  the  fact  that 
the  eyes  become  inured  to  labor  like  the  other  organs  of  the  body  by 
judicious  exercise.  It  is  also  due  to  the  fact  that  but  very  slight  ac- 
commodation is  required  of  the  eyes,  the  eyes  remaining  nearly  in  a 
condition  of  rest  as  for  distant  objects.  The  fatigue  incident  upon 


62 


LIGHTING  AND  FOCUSING 


{_CH. 


using  the  microscope  at  first  is  due  partly  at  least  to  the  constant  effort 
on  the  part  of  the  observer  to  remedy  the  defects  of  focusing  the 
microscope  by  accommodation  of  the  eyes.  This  should  be  avoided 
and  the  fine  adjustment  of  the  microscope  used  instead  of  the  muscles 
of  accommodation.  With  a  microscope  of  the  best  quality,  and  suita- 
ble light — that  is  light  which  is  steady  and  not  so  bright  as  to  dazzle 
the  eyes  nor  so  dim  as  to  strain  them  in  determining  details — micro- 
scopic work  should  improve  rather  than  injure  the  sight. 


7  X  44     cm. 


FIG.  60.  Double  Eye  Shade.  This  is 
readily  made  by  taking  some  thick  bris- 
tol  board  7x14  centimeters  and  making 
an  oblong  opening  with  rounded  ends 
(o-o)  and  of  such  a  diameter  that  it  goes 
readily  over  the  tube  of  the  microscope. 
This  is  then  covered  on  both  sides  with 
velveteen  and  a  central  slit  (s)  made  in 

^ </      the  cloth.      This  admits  the  tube  of  the 

microscope  and  holds  the  screen  in  posi- 
tion. It  may  readily  be  pulled  from  side  to  side  and  thus  serves  for  either  eye,  or 
for  the  use  of  the  eyes  alternately. 


FIG.  60  a.  Adjusting  Eye-Shade.  This  is  prepared  like  the  preceding  by  cov- 
ering a  card  about  6  x  12  centimeters  with  black  velveteen.  A  copper  wire  about 
1  mm.(l/&  in. )  and  of  the  right  length  is  curved  as  shown  in  the  figure.  Its  ends  are 
rounded,  and  finally  it  is  put  under  the  cloth  and  sewed  carefully  all  around.  The 
card  and  cloth  are  then  cut  as  shown.  The  flexible  wire  makes  it  possible  to  put 
the  screen  on  the  tube  at  any  level. 

§  117.  Position  and  Character  of  the  'Work-Table. — The 
work-table  should  be  very  firm  and  large  (60 x  120  cm.;  24x48  in.), 
so  that  the  necessary  apparatus  and  material  for  work  may  not  be  too 
crowded.  The  table  should  also  be  of  the  right  height  to  make  work 
by  it  comfortable.  An  adjustable  stool,  something  like  a  piano  stool  is 
convenient,  then  one  may  vary  the  height  corresponding  to  the  neces- 


CH.  If]  LIGHTING  AND  FOCUSING  63 

sities  of  special  cases.  It  is  a  great  advantage  to  sit  facing  the  window 
if  daylight  is  used,  then  the  hands  do  not  constantly  interfere  with 
the  illumination.  To  avoid  the  discomfort  of  facing  the  light  a  screen 
like  that  shown  in  Fig.  59  is  very  useful  (see  also  under  lighting, 

§62). 

TESTING   THE    MICROSCOPE 

|  118.  Testing  the  Microscope. — To  be  of  real  value  this  must  be  accom- 
plished by  a  person  with  both  theoretical  and  practical  knowledge,  and  also  with 
an  unprejudiced  mind.  Such  a  person  is  not  common,  and  when  found  does  not 
show  over  anxiety  to  pass  judgment.  Those  most  ready  to  offer  advice  should 
as  a  rule  be  avoided,  for  in  most  cases  they  simply  "have  an  ax  to  grind,"  and  are 
sure  to  commend  only  those  instruments  that  conform  to  the  "fad"  of  the  day. 
From  the  writer's  experience  is  seems  safe  to  say  that  the  inexperienced  can  do  no 
better  than  to  state  clearly  what  he  wishes  to  do  with  a  microscope  and  then  trust 
to  the  judgment  of  one  of  the  optical  companies.  The  makers  of  microscopes  and 
objectives  guard  with  jealous  care  the  excellence  of  both  the  mechanical  and 
optical  part  of  their  work,  and  send  out  only  instruments  that  have  been  carefully 
tested  and  found  to  conform  to  the  standard.  This  would  be  done'  as  a  matter  of 
business  prudence  on  their  part,  but  it  is  believed  by  the  writer  that  microscope 
makers  are  artists  first  and  take  an  artist's  pride  in  their  work,  they  therefore  have 
a  stimulus  to  excellence  greater  than  business  prudence  alone  could  give. 

2  119.  Mechanical  Parts. — All  of  the  parts  should  be  firm,  and  not  too  easily 
shaken.  Bearings  should  work  smoothly.  The  mirror  should  remain  in  any 
position  in  which  it  is  placed. 

Focusing  Adjustments. — The  coarse  or  rapid  adjustment  should  be  by  rack 
and  pinion,  and  work  so  smoothly  that  even  the  highest  power  can  be  easily  focused 
with  it.  In  no  case  should  it  work  so  easily  that  the  body  of  the  microscope  is 
liable  to  run  down  and  plunge  the  objective  into  the  object.  If  any  of  the  above 
defects  appear  in  a  microscope  that  has  been  used  for  some  time,  a  person  with 
modjerate  mechanical  instinct  will  be  able  to  tighten  the  proper  screw,  etc. 

The  Fine  Adjustment  is  more  difficult  to  deal  with.  From  the  nature  of  its 
purpose  unless  it  is  approximately  perfect,  it  would  be  better  off  the  microscope 
entirely.  It  has  been  much  improved  recently. 

It  should  work  smoothly  and  be  so  balanced  that  one  cannot  tell  by  the  feel- 
ing when  using  it  whether  the  screw  is  going  up  or  down.  Then  there  should  be 
absolutely  no  motion  except  in  the  direction  of  the  optic  axis,  otherwise  the 
image  will  appear  to  sway  even  with  central  light.  Compare  the  appearance 
when  using  the  coarse  and  when  using  the  fine  adjustment.  There  should  be  no 
swaying  of  the  image  with  either  if  the  light  is  central  ($  77). 

|  120.  Testing  the  Optical  Parts. — As  stated  in  the  beginning,  this  can  be 
done  satisfactorily  only  by  an  expert  judge.  It  would  be  of  very  great  advantage 
to  the  student  if  he  could  have  the  help  of  such  a  person.  In  no  case  is  a  micro- 
scope to  be  condemned  by  an  inexperienced  person.  If  the  beginner  will  bear  in 
mind  that  his  failures  are  due  mostly  to  his  own  lack  of  knowledge  and  lack  of 
skill ;  and  will  truly  endeavor  to  learn  and  apply  the  principles  laid  down  in  this 
and  in  the  standard  works  referred  to,  he  will  learn  after  a  while  to  estimate  at 
their  true  value  all  the  pieces  of  his  microscope.  (See  Ch.  X). 


64  LA  BORA  TOR  Y  MICROSCOPES  \_CH.  II 

LABORATORY   AND    HIGH-SCHOOL   COMPOUND 
MICROSCOPES 


$  121.  Optical  Parts. — A  great  deal  of  beginning  work  with  the  microscope  in 
biological  laboratories  is  done  with  simple  and  inexpensive  apparatus.  Indeed  if 
one  contemplates  the  large  classes  in  the  high  schools,  the  universities  and  med- 
ical schools,  it  can  be  readily  understood  that  microscopes  costing  from  $25-50  each 
and  magnifying  from  25  to  500  diameters,  are  all  that  can  be  expected.  But  for 
the  purpose  of  modern  histological  investigation  and  of  advanced  microscopical 
work  in  general,  a  microscope  should  have  something  like  the  following  character  : 
Its  optical  outfit  should  comprise,  (a)  dry  objectives  of  50  mm. (2  in.),  16-18  mm. 
( %  in. )  and  3  mm.  ( %  in. )  equivalent  focus.  There  should  be  present  also  a 
2  mm.  (^2  in. )  °r  :-5  nitn.  (T^  in. )  homogeneous  immersion  objective.  Of  oculars 
there  should  be  several  of  different  power.  A  centering  substage  condenser, 
and  an  Abbe  camera  lucida  are  also  necessities,  and  a  micro-spectroscope  and  a 
micro-polarizer  are  very  desirable. 

Even  in  case  all  the  optical  parts  cannot  be  obtained  in  the  beginning,  it  is 
wise  to  secure  a  stand  upon  which  all  may  be  used  when  they  are  finally  secured. 

As  to  the  objectives.  The  best  that  can  be  afforded  should  be  obtained.  Cer- 
tainly at  the  present,  the  apochromatics  stand  at  the  head,  although  the  best 
achromatic  objectives  approach  them  very  closely. 

|  122.  Mechanical  Parts  or  Stand. — The  stand  should  be  low  enongh  so  that 
it  can  be  used  in  a  vertical  position  on  an  ordinary  table  without  inconvenience  ; 
it  should  have  a  jointed  (flexible)  pillar  for  inclination  at  any  angle  to  the  hori- 
zontal. The  adjustments  for  focusing  should  be  two, — a  coarse  adjustment  or 
rapid  movement  with  rack  and  pinion,  and  a  fine  adjustment  by  means  of  a  mi- 
crometer screw.  Both  adjustments  should  move  the  entire  tube  of  the  microscope. 
The  body  or  tube  should  be  short  enough  for  objectives  corrected  for  the  short  or 
160  millimeter  tube-length.  It  is  an  advantage  to  have  the  draw-tube  graduated 
in  centimeters  and  millimeters.  The  lower  end  of  the  draw  tube  and  of  the  tube 
should  each  possess  a  standard  screw  for  objectives  (frontispiece).  The  stage 
should  be  quite  large  for  the  examination  of  slides  with  serial  sections  and  other 
large  objects.  The  substage  fittings  should  be  so  arranged  as  to  enable  one  to  use 
the  .condenser  or  to  dispense  entirely  with  diaphragms.  The  condenser  mount- 
ing should  allow  up  and  down  motion. 


STANDARD   SIZES   RECOMMENDED    BY   THE   ROYAL 
MICROSCOPICAL    SOCIETY 


§  123.  Society  Screw. — Owing  to  the  lack  of  uniformity  in  screws  for  micro- 
scope objectives,  the  Royal  Microscopical  Society  of  London,  in  1857,  made  an 
earnest  effort  to  introduce  a  standard  size.  The  specifications  of  this  standard  are 


CH.  //]  LABORATORY  MICROSCOPES  65 

as  follows  :  "Whitworth  thread,  i.  e.,  a  V  shaped  thread,  sides  of  thread  inclined 
to  angle  of  55°  to  each  other,  one-sixth  of  the  V  depth  of  the  thread  being 
rounded  off  at  the  top  of  the  thread,  and  one-sixth  of  the  thread  being  rounded  off 
at  the  bottom  of  the  thread.  Pitch  of  screw,  36  to  the  inch  ;  length  of  thread  on 
object-glass,  0.125  inch  ;  plain  fitting  above  thread  of  object-glass,  0.15  inch  long, 
to  be  about  the  size  of  the  bottom  of  male  thread  ;  length  of  thread  of  nose-piece 
[on  the  lower  end  of  the  tube  of  the  microscope] ,  not  less  than  o.  25  inch  ;  diam- 
eter of  the  object-glass  screw  at  the  bottom  of  the  screw,  0.7626  inch  ;  diameter 
of  the  nose-piece  screw  at  the  bottom  of  the  thread,  0.8  inch." 

In  order  to  facilitate  the  introduction  of  this  universal  screw,  or  as  it  soon 
came  to  be  called  "  The  Society  Screw,"  the  Royal  Microscopical  Society  undertook 
to  supply  standard  taps.  From  the  mechanical  difficulty  in  making  these  taps 
perfect  there  soon  came  to  be  considerable  difference  in  the  "Society  Screws, "  and 
the  object  of  the  society  in  providing  a  universal  screw  was  partly  defeated. 

In  1884  the  American  Microscopical  Society  appointed  Mr.  Edward  Bausch 
and  Prof.  William  A.  Rogers  upon  a  committee  to  correspond  with  the  Royal 
Microscopical  Society,  with  a  view  to  perfecting  the  standard  "Society  Screw," 
or  of  adopting  another  standard  and  of  perfecting  methods  by  which  the  screws  of 
all  makers  might  be  truly  uniform.  Although  this  matter  was  earnestly  consid- 
ered at  the  time  by  the  Royal  Microscopical  Society,  the  mechanical  difficulties 
were  so  great  that  the  improvements  were  abandoned. 

Fortunately,  however,  during  the  year  (1896)  that  society  again  took  hold  of 
the  matter  in  earnest,  and  the  "Society  Screw"  is  now  accurate,  and  facilities  for 
obtaining  the  standard  are  so  good  that  there  is  a  reasonable  certainty  that  the 
universal  screw  for  microscopic  objectives  may  be  realized.  It  is  astonishing  to 
see  how  widely  the  "Society  Screw"  has  been  adopted.  Indeed  there  is  not  a 
maker  of  first-class  microscopes  in  the  world  who  does  not  supply  the  objectives 
and  stands  with  the  "Society  Screw, "  and  an  objective  in  England  or  America  which 
does  not  have  this  screw  should  be  looked  upon  with  suspicion.  That  is,  it  is 
either  old,  cheap,  or  not  the  product  of  one  of  the  great  opticians.  For  the  Stand- 
ard, or  "Society  Screw,"  see  :  Trans.  Roy.  Micr.  Soc. ,  1857,  pp.  39-41  ;  1859,  pp. 
92-97  ;  1860,  pp.  103-104.  (All  to  be  found  in  Quar.  Jour.  Micr.  Sci.,  o.  s.,  vols. 
VI,  VII  and  VIII).  Proc.  Amer.  Micr.  Soc.  1884,  p.  274;  1886,  p.  199  ;  1893,  p. 
38.  Journal  of  the  Royal  Microscopical  Society,  August,  1896. 

In  this  last  paper  of  four  pages  the  matter  is  very  carefully  gone  over  and  full 
specifications  of  the  new  screw  given.  It  conforms  almost  exactly  with  the  orig- 
inal standard  adopted  by  the  society,  but  means  have  been  devised  by  which  it 
may  be  kept  standard. 

§124.  Standard  Size  Oculars  and  Substage  Condensers. — For  a  considera- 
tion of  these,  with  measurements,  see  f  46,  87. 


MARKERS    AND    MECHANICAL   STAGES 


Markers  are  devices  to  facilitate  the  finding  of  some  object  or  part  which  it  is 
especially  desired  to  refer  to  again  or  to  demonstrate  to  a  class.  The  mechanical 
stage  makes  it  much  easier  to  follow  out  a  series  of  objects,  to  move  the  slide 
when  using  high  powers,  and  for  complete  exploration  of  a  preparation.  Most  of 
the  mechanical  stages  have  scales  or  scales  and  verniers  by  which  an  object  once 
recorded  may  be  readily  found  again. 


66 


LABORATORY  MICROSCOPES 


{_CH. 


\  125.  Marker  for  Preparations.  (Figs.  61-66). — This  instrument  consists  of  an 
objective-like  attachment  which  may  be  screwed  into  the  nose-piece  of  the  micro- 
scope. It  bears  on  its  lower  end  (Figs.  61-3)  a  small  brush  and  the  brush  can  be 
made  more  or  less  eccentric  and  can  be  rotated,  thus  making  a  larger  or  smaller 
circle.  In  using  the  marker  the  brush  is  dipped  in  colored  shellac  or  other  cement 
and  when  the  part  of  the  preparation  to  be  marked  is  found  and  put  exactly  in  the 
middle  of  the  field  the  objective  is  turned  aside  and  the  marker  turned  into  posi- 
tion. The  brush  is  brought  carefully  in  contact  with  the  cover-glass  and  rotated. 
This  will  make  a  delicate  ring  of  the  colored  cement  around  the  object.  Within 
this  very  small  area  the  desired  object  can  be  easily  found  on  any  microscope. 
The  brush  of  the  marker  should  be  cleaned  with  95%  alcohol  after  it  is  used. 
(Proc.  Amer.  Micr.  Soc.,  1894,  pp.  112-118). 


61. 


62. 


63- 


FIGS.  61-63.     Sectional  Views  of  the  two  Forms  of  the  Marker. 

FIG.  61.  The  simplest  form  of  marker.  It  consists  of  the  part  SS  with  the 
milled  edge  (M}.  This  part  bears  the  society  or  objective  screw  for  attaching  the 
marker  to  the  microscope.  R.  Rotating  part  of  the  marker.  This  bears  the  eccen- 
tric brush  (B}  at  its  lower  end.  The  brush  is  on  a  wire  (  W).  This  wire  is  eccen- 
tric, and  may  be  made  more  or  less  so  by  bending  the  wire.  The  central  dotted 
line  coincides  with  the  axis  of  the  microscope.  The  revolving  part  is  connected 
with  the  ''Society  Screw"  by  the  small  screw  (S }. 

FIG.  62.  61S,  R,  and  B.  All  parts  same  as  with  Fig.  6s,  except  that  the  brush 
is  carried  by  a  sliding  cylinder  the  end  view  being  indicated  in  Fig.  <5j. 


CH.  //] 


LABORATORY  MICROSCOPES 


67 


66 

FIGS.  64,  65,  66.     Specimens  Showing  the  Use  of  the  Marker. 

In  Fig.  64  a  section  of  a  series  is  marked  to  indicate  that  this  section  shows  some- 
thing especially  well.  In  Fig.  65  some  blood  corpuscles  showing  ingested  carbon 
very  satisfactorily  are  surrounded  by  a  minute  ring,  and  in  Fig.  66  the  lines  of  a 
micrometer  are  ringed  to  facilitate  finding  the  lines. 

\  126.  Pointer  in  the  Ocular. — The  Germans  have  a  pointer  ocular  (Spitzen- 
Okular),  an  ocular  with  one  or  two  delicate  rods  or  pointers  at  the  level  of  the 
real  image,  that  is,  at  the  level  of  the  diaphragm  (Figs.  21,  30  D).  For  the  pur- 
poses of  demonstrating  any  particular  structure  or  object  in  the  field,  a  temporary 
pointer  may  be  easily  inserted  in  any  ocular  as  follows  :  Remove  the  eye-lens  and 
with  a  little  mucilage  or  Canada  Balsam  fasten  an  eye-lash  (cilium)  to  the  diaphragm 
(Fig.  30  D)  so  that  it  will  project  about  half  way  across  the  opening.  If  one  uses 
this  ocular,  the  pointer  will  appear  in  the  field  and  one  can  place  the  specimen  so 
that  the  pointer  indicates  it  exactly,  as  in  using  a  pointer  on  a  diagram  or  on  the 
black-board.  It  is  not  known  to  the  author  wrho  devised  this  method.  It  is  cer- 
tainly of  the  greatest  advantage  in  demonstrating  objects  like  amoebas  or  white 
blood  corpuscles  to  persons  not  familiar  with  them,  as  the  field  is  liable  to  have  in 
it  many  other  objects  which  are  more  easily  seen. 

\  127.  Mechanical  Stage. — For  High  School  and  ordinary  laboratory  work  a 
mechanical  stage  is  not  needed  ;  but  for  much  work,  especially  where  high  objec- 
tives are  used  a  mechanical  stage  is  of  great  advantage.  It  is  also  advantageous  if 
the  mechanical  stage  can  be  easily  removed  (see  Figs.  67  to  70).  The  one  found 
on  the  most  expensive  American  and  English  microscopes  for  the  last  twenty  years 
and  the  one  now  present  on  the  larger  continental  microscopes,  is  excellent  for 
high  powers  and  preparations  of  moderate  dimensions,  but  for  the  study  of  serial 
sections  and  large  sections  or  preparations  in  general,  mechanical  stages  like  those 
shown  in  Figs.  68-69  are  more  useful.  This  form  of  mechanical  stage  has  the 
advantage  of  giving  great  lateral  and  forward  and  backward  motion.  It  is  a  mod- 
ification of  the  mechanical  stage  of  Tolles.  The  modification  consists  in  doing 
away  with  the  thin  plate  and  having  a  clamp  to  catch  the  ends  of  the  glass  slide. 
The  slide  is  then  moved  on  the  face  of  the  stage  proper.  This  modification  was 
first  made  by  Mayall.  It  has  since  been  modified  by  Reichert,  Zeiss,  Leitz,  and 
others  in  Europe  and  by  the  Bausch  &  Lomb  Optical  Co. ,  Queen  &  Co. ,  and  the 
Spencer  Lens  Co.,  in  America. — Jour.  Roy.  Micr.  Soc.,  1885,  p.  122.  See  also 
Zeit.  Wiss.  Mikroskopie  ( II) ,  1885,  pp.  289-295  ;  1887  (IV,  pp.  25-30). 

Those  figured  below  have  the  great  advantage  of  ready  removal  from  the  stage 
of  the  microscope,  thus  leaving  it  free.  They  have  also  the  very  excellent  feature 
that  with  them  one  can  explore  an  entire  slide  full  of  serial  sections,  as  the  sec- 
tions are  ordinarily  mounted,  i.  e.,  under  a  cover-glass  24X50  mm. 


68 


LA  BORA  TOR  Y  MICROSCOPES 


\CH.  II 


FIG 


FIGS.  67,  6ya.  The  removable  mechanical  stage  of  the  Bausch  &  Lomb  Opti- 
cal Company.  In  the  upper  figure  it  is  in  position  on  the  stage  of  the  microscope  ; 
in  the  lower  figure  only  the  clamping  part  is  in  position,  the  rest  having  been 
removed  to  leave  the  stage  of  the  microscope  free. 


CH.  II] 


LA  BORA  TOR  Y  MICROSCOPES 


69 


FIG.  68. 


FIG.  68a. 


FIG.  68,  68a.  Two  forms  of  removable  mechanical  stage  by  Leitz.  68  is  some- 
what more  complex  and  expensive.  Both  have  the  desirable  features  mentioned 
in  \  127. 


yo 


LABORATORY  MICROSCOPES 


[CH.  II 


FIG.  69.  The  removable  mechanical  stage  of  the  Spencer  Lens  Co.  It  is1  a 
modification  of  a  form  devised  by  Winkel.  Besides  the  general  features  mentioned 
in  \.  127  it  has  the  advantage  of  fitting  any  square  stage.  It  is  fastened  to  the  stage 
by  the  clamps  shown  at  the  right.  Another  form,  is  made  having  one  screw  on 
the  side. 


CH.  //]  LABORATORY  MICROSCOPES  71 


FIG.  70.  Krauss1  Method  of 
Marking  Objectives  on  a  Revolving 
Nose- Piece. 

As  seen  in  the  figure,  the  equiv- 
alent focus  of  the  objective  is  en- 
graved on  the  diaphragm  above  the 
back  lens  and  may  be  very  readily 
seen  in  rotating  the  nose-piece.  This 
is  of  great  advantage,  as  one  can  see 
what  objective  is  coming  into  place 
without  trouble.  It  is  also  an  ad- 
vantage in  showing  where  each  ob- 
jective belongs  when  the  microscope 
comes  from  the  ma n  ufacturers.  The 
method  is  coming  into  general  use. 


FIGURES  OF  LABORATORY  AND  HIGH  SCHOOL 
MICROSCOPES 


In  order  that  teachers  and  students  may  get  a  good  general  idea  of  the  appear- 
ance of  microscopes  of  various  makers  for  high  school  and  advanced  laboratory 
work  a  few  pictures  are  appended  of  the  microscopes  most  used  in  the  United 
States.  This  has  been  rendered  possible  by  the  courtesy  of  the  manufacturers  or 
importers.  The  microscopes  are  arranged  in  alphabetical  order  of  the  makers. 

Laboratory  microscopes  which  will  answer  nearly  all  the  requirements  for 
work  in  Biology,  including  Histology,  Embryology,  Pathology  and  Bacteriology, 
are  listed  in  the  makers  catalogs  at  about  $100.00.  The  less  expensive  micro- 
scopes shown  are  listed  at  $25  to  $45.  There  is  usually  a  discount  of  10%  or  more 
from  these  prices.  Fortunately  in  the  State  of  New  York  the  State  pays  half  for 
high  school  apparatus,  so  that  there  is  no  reason  why  every  high  school  should 
not  be  properly  equipped  with  microscopes  of  a  good  grade.  To  avoid  misunder- 
standing it  should  be  added  that  the  quality  of  the  oculars  and  objectives  on  the 
high  school  microscopes  figured  is  the  same  as  for  the  best  laboratory  micro- 
scopes. The  mechanical  work  also  is  of  excellent  quality. 

During  the  last  ten  years  great  vigor  has  been  shown  in  the  microscopical 
world.  This  has  been  stimulated  largely  by  the  activity  in  biological  science  and 
the  widespread  appreciation  of  the  microscope,  not  only  as  a  desirable,  but  as  a 
necessary  instrument  for  study  and  research.  The  production  of  the  new  kinds 
of  glass,  (Jena  glass),  and  the  apochromatic  objectives  has  been  a  no  less  potent 
factor  in  promoting  progress.  The  student  is  advised  to  write  to  one  or  more  of 
the  opticians  for  complete  catalogs.  (See  list,  p.  2  of  cover). 


LABORATORY  MICROSCOPES 


[CJL   II 


PATENT   APPLIED    FOR 


FIG.  71.  The  new  cone  movement,  fine  adjustment  for  the  Photo-Micrographic 
and  the  Nos.  20  and  35  Microscopes  of  the  Spencer  Lens  Co.  See  the  figures  of  those 
microscopes  pp.  82-87. 


FIG.  71  a.     Capped  balsam  bottle  of  WhitalL  Tatum  &  Co.     This  is  far  more 
satisfactory  than  the  small  spirit  lamp  figured  on  p.  172. 


Bausch  &  Lomb  Continental  Type  Microscope  BB. 

Fig.  //. — The  most  common  type  in  American  University  Laboratories. 


Bausch  &  Lomb  Continental  Type  Microscope  CA. 

Fig.  72. — Especially  adapted  for  Bacteriological  and 
other   work   requiring  large    stage   room. 


Bausch  &  Lomb  Continental  Type  Microscope  DD. 

f&  73- — This  instrument  is  adapted  for  every  kind  of  work,  especially 
critical  Cytological.  Bacteriological  and  Photomicrographic  studies. 


Bausch  &  Lomb  Petrographical   Microscope  LA. 

Fig.  /jvz. — Specially  designed  throughout  for  Petrography. 


Bausch   &   Lomb   Continental   Microscope  AC. 

Fig.  fjb. — A  simple  construction,  very  durable  and  low  in  price. 


Bausch  &  Lomb   Continental    Type  Microscope  B. 

A  type  adapted  to  High  School  work  when  a  low-priced  instrument 

is  required. 


CH.  //] 


LABORATORY  MICROSCOPES 


75 


FIG.  74.     R.  &  J.  Beck's  New  Continental  Microscope,  No.  1125  ( Williams, 
Brown  &  Earle,  Philadelphia}. 


76 


LABOR  A  TOR  Y  MICROSCOPES 


[_CH. 


FIG.  75.     E.  Leitz  Microscope  II  C.     (  Wm.  Krafft,  New  York}. 


FIG.  75  a.     The  new  microscope  of  Leitz  -with  large  stage  space,  large  tube 
and  new  form  fine  adjustment  (see  over}. 


FIGS.  75,  b,  c.  New  Fine  Adjustment  of  Leitz.  The  movement  is  produced 
by  a  cam  rotating  on  a  fixed  axis  (/).  The  cam  is  moved  by  a  worm  wheel  (d) 
which  engages  the  thread  of  an  endless  screw  (a]  from  two  sides  (Fig.  75). 

One  complete  turn  of  the  endless  screw  (a]  produces  an  adjustment  of  i-ioth 
mm.  By  means  of  the  graduated  drum  (r),  on  the  axis  of  the  screw,  one  can 
easily  read  i-iooth  of  a  revolution  and  thus  determine  an  adjustment  of  i-ioooth 
mm.  Cuts  loaned  by  Wm.  Krafft,  N.  Y. 


CH.  II  ] 


LABORATORY  MICROSCOPES 


77 


FIG.  76.     Leitz  Microscope  HE.  It  will  be  noticed  that  this  microscope  has  no 
joint  for  inclination  (  Wm.  Krafft,  N.  Y. ). 


LABORATORY  MICROSCOPES 


[CfJ.  II 


FIG.  77.  NacheVs  Microscope  6  bis.  Old  model.  Nachet  is  a  successor  to 
Hartnack  who  introduced  the  present  "Continental  Model'1'1  May  all,  Cantor  Lec- 
tures, p.  68  (Franklin  Laboratory  Supply  Co.,  Boston}. 


CH.  //] 


LABORATORY  MICROSCOPES 


79 


FIG.  78.     NacheVs  Microscope,  No.  u  (Franklin  Laboratory  Supply  Co.,  Boston). 

This  microscope  has  no  joint  for  inclination,  and  no  rack  and  pinion  for  coarse 
adjustment.  For  coarse  adjustment,  the  tube  is  pulled  up  and  down  with  the  hands. 
This  kind  of  coarse  adjustment  was  much  more  common  ten  years  ago  than  now. 
(See  also  Fig.  82. ) 


8o 


LABOR  A  TOR  Y  MICROSCOPES 


[CH.  II 


FIG.  79.  A.  Queen  &  Co's  Continental  Microscope ,  No.  II.  B.  Dust-prooj, 
triple  nose-piece.  The  difference  between  this  and  the  ordinary  form  can  be  seen 
by  comparing  with  Fig.  36.  This  form  of  revolving  nose-piece  has  been  made  for 
many  years  by  Winkel  of  Goettingen. 


CH.  77] 


LABORATORY  MICROSCOPES 


81 


FIG.  80.     Queen  &  Co's  Acme  Microscope  for  Schools. 


82 


LABORATORY  MICROSCOPES 


\_CH. 


FIG.  81.  Reichert's  New  Mi- 
croscope, No.  Ill  B.  (Richards 
&  Co.,  N.  Y.  and  Chicago}. 


Fig.  82.     The  Spencer  Lens  Company's  Microscope  No.   1. 

1903  Model. 


Fig.  83.     The  Spencer  L,ens  Company's  Microscope  No.   2. 
general  science  work  in  college  and  high  school 
laboratories.     Latest  model. 


For 


Fig.  84.     The  Spencer  Lens  Company's  Microscope  No.  5.     For 
professional  and  laboratory  use. 


Fig.  85.     The  Spencer  Lens  Company's  Microscope  No.  6. 
daily  designed  for  photo-micrography,  but  equally 
well  suited  for  ordinary  work. 


Espe- 


The  Spencer  Lens  Company's  New  No.  4  Stand,  with  special  fine 
adjustment  and  handle  for  carrying. 


Patented  June  24,  1902. 


Fig.  85a.  The  Spencer  L,ens  Company's  New  Attachable 
Mechanical  Stage.  Both  movements  may  be  operated  by  the 
fingers  of  one  hand,  the  milled  heads  being  placed  together  at  one 
end  with  coincident  axes.  At  the  other  end  is  a  milled  head  for 
the  lateral  movement  so  that  either  hand  may  be  used  therefor. 
The  center  guide  screw  furnishes  a  stop  for  the  slide  carrier  so 
that  the  objective  cannot  be  injured, 


CH.  //] 


LABORATORY  MICROSCOPES 


FIG.  86.  Zeiss  Microscope  /a  with  Mechanical  Stage.  This  figure  from  Zeiss' 
Catalog  No.  30,  represents  the  Continental  Model  of  Microscope  in  its  most  perfect 
form. 

K.     Milled  head  of  the  screw  for  the  lateral  movements  of  the  stage. 

L.     Screw  for  fixing  the  laterally  moving  mechanism  of  the  stage.     By  un- 
screwing this  the  laterally  moving  part  may  be  removed,  leaving  the  plain  stage. 
W.     Screw  for  moving  the  stage  forward  and  backward. 


88 


LABOR  A  TOR  Y  MICROSCOPES 


[CH.  II 


FIG.  87,     Zcntmeyer's  Microscope,  A7o.  V. 


CH.  //] 


LA  BORA  TOR  Y  MICROSCOPES 


89 


FIG.  88.     Zentmayer's  Microscope,  Xo.  IV. 


CHAPTER   III 


INTERPRETATION    OF    APPEARANCES 


APPARATUS   AND    MATERIAL   FOR    CHAPTER    III 

A  laboratory,  compound  microscope  (  \  121 );  Preparation  of  fly's  wing  ;  50  per 
cent,  glycerin;  Slides  and  covers;  Preparation  of  letters  in  stairs  (Fig.  89); 
Mucilage  for  air-bubbles  and  olive  or  clove  oil  for  oil-globules  (\  136-139).  Solid 
glass  rod,  and  glass  tube  (\  144-146);  Collodion  ($  146);  Carmine,  India  ink,  or 
lamp  black  (§  148-150);  Frog,  castor  oil  and  micro-polariscope  (§  152). 

INTERPRETATION   OF   APPEARANCES    UNDER    THE   MICROSCOPE 

§  129.  General  Remarks. — The  experiments  in  this  chapter  are 
given  secondarily  for  drill  in  manipulation,  but  primarily  so  that  the 
student  may  not  be  led  into  error  or  be  puzzled  by  appearances  which 
are  constantly  met  with  in  microscopical  investigation.  Anyone  can 
look  into  a  microscope,  but  it  is  quite  another  matter  to  interpret  cor- 
rectly the  meaning  of  the  appearances  seen. 

It  is  especially  important  to  remember  that  the  more  of  the  relations 
of  any  object  are  known,  the  truer  is  the  comprehension  of  the  object. 
In  microscopical  investigation  every  object  should  be  scrutinized  from 
all  sides  and  under  all  conditions  in  which  it  is  likely. to  occur  in  nature 
and  in  microscopical  investigation.  It  is  best  also  to  begin  with  objects 
of  considerable  size  whose  character  is  well  known,  to  look  at  them 
carefully  with  the  unaided  eye  so  as  to  see  them  as  wholes  and  in  their 
natural  setting  ;  then  a  low  power  is  used,  and  so  on,  step  by  step  until 
the  highest  power  available  has  been  employed.  One  will  in  this  way 
see  less  and  less  of  the  object  as  a  whole,  but  every  increase  in  magnifi- 
cation will  give  increased  prominence  to  detail,  detail  which  might  be 
meaningless  when  taken  alone  and  independent  of  the  object  as  a 
whole.  The  pertinence  of  this  advice  will  be  appreciated  when  the 
student  undertakes  to  solve  the  problems  of  histology  ;  for  even  after  all 
the  years  of  incessant  labor  spent  in  trying  to  make  out  the  structure 
of  man  and  the  lower  animals,  many  details  are  still  in  doubt,  the 
same  visual  appearances  being  quite  differently  interpreted  by  eminent 
observers. 


CH.  Ill]  INTERPRETATION  OF  APPEARANCES  91 

Appearances  which  seem  perfectly  unmistakable  with  a  low  power 
may  be  found  erroneous  or  very  inadequate,  for  details  of  structure  that 
were  indistinguishable  with  the  low  power  may  become  perfectly  evi- 
dent with  a  higher  power  or  a  more  perfect  objective.  Indeed  the  prob- 
lems of  microscopic  structure  appear  to  become  ever  more  complex,  for 
difficulties  overcome  by  improvements  in  the  microscope  simply  give 
place  to  new  difficulties,  which  in  some  cases  render  the  subject  more 
obscure  than  it  appeared  to  be  with  the  less  perfect  appliances. 

The  need  of  the  most  careful  observation  and  constant  watchful- 
ness lest  the  appearances  may  be  deceptive  are  thus  admirably  stated 
by  Dallinger  (see  Carpenter-Dallinger,  p.  427):  "The  correctness  of 
the  conclusions  which  the  microscopist  will  draw  regarding  the  nature 
of  any  object  from  the  visual  appearances  which  it  presents  to  him 
when  examined  in  the  various  modes  now  specified  will  necessarily 
depend  in  a  great  degree  upon  his  previous  experience  in  microscopic 
observation  and  upon  his  knowledge  of  the  class  of  bodies  to  which 
the  particular  specimen  may  belong.  Not  only  are  observations  of 
any  kind  liable  to  certain  fallacies  arising  out  of  the  previous  notions 
which  the  observer  may  entertain  in  regard  to  the  constitution  of  the 
objects  or  the  nature  of  the  actions  to  which  his  attention  is  directed, 
but  even  the  most  practiced  observer  is  apt  to  take  no  note  of  such 
phenomena  as  his  mind  is  not  prepared  to  appreciate.  Errors  and  im- 
perfections of  this  kind  can  only  be  corrected,  it  is  obvious,  by  general 
advance  in  scientific  knowledge  ;  but  the  history  of  them  affords  a  use- 
ful warning  against  hasty  conclusions  drawn  from  a  too  cursory  exam- 
ination. If  the  history  of  almost  any  scientific  investigation  were 
fully  made  known  it  would  generally  appear  that  the  stability  and 
completeness  of  the  conclusions  finally  arrived  at  had  been  only 
attained  after  many  modifications,  or  even  entire  alterations,  of  doctrine. 
And  it  is  therefore  of  such  great  importance  as  to  be  almost  essential 
to  the  correctness  of  our  conclusions  that  they  should  not  be  finally 
formed  and  announced  until  they  have  been  tested  in  every  conceivable 
mode.  It  is  due  to  science  that  it  should  be  burdened  with  as  few  false 
facts  [artifacts]  and  false  doctrines  as  possible.  It  is  due  to  other 
truth-seekers  that  they  should  not  be  misled,  to  the  great  waste  of 
their  time  and  pains,  by  our  errors.  And  it  is  due  to  ourselves  that 
we  should  not  commit  our  reputation  to  the  chance  of  impairment  by 
the  premature  formation  and  publication  of  conclusions  which  may  be 
at  once  reversed  by  other  observers  better  informed  than  ourselves,  or 
may  be  proved  fallacious  at  some  future  time,  perhaps  even  by  our 


92  INTERPRETATION  OF  APPEARANCES  [Cff.  Ill 

own  more  extended  and  careful  researches.  The  suspension  of  the  judg- 
ment whenever  there  seems  room  for  doubt  is  a  lesson  inculcated  by  all 
those  philosophers  who  have  gained  the  highest  repute  for  practical 
wisdom  ;  and  it  is  one  which  the  microscopist  cannot  too  soon  learn  or 
too  constantly  practice." 

For  these  experiments  no  condenser  is  to  be  used  except  where 
specifically  indicated. 

§  130.  Dust  or  Cloudiness  on  the  Ocular. — Employ  the  16 
mm.  (2/i  in.)  objective,  low  ocular,  and  fly's  wing  as  object. 

Unscrew  the  field-lens  and  put  some  particles  of  lint  from  dark 
cloth  on  its  upper  surface.  Replace  the  field-lens  and  put  the  ocular 
in  position  (§  48).  Light  the  field  well  and  focus  sharply.  The  im- 
age will  be  clear,  but  part  of  the  field  will  be  obscured  by  the  irregular 
outline  of  the  particles  of  lint.  Move  the  object  to  make  sure  this 
appearance  is  not  due  to  it. 

Grasp  the  ocular  by  the  milled  ring,  just  above  the  tube  of  the 
microscope,  and  rotate  it.  The  irregular  objects  will  rotate  with  the 
ocular.  Cloudiness  or  particles  of  dust  on  any  part  of  the  ocular  may 
be  detected  in  this  way. 

§  131.  Dust  or  Cloudiness  on  the  Objective. — Employ  the 
same  ocular  and  objective  as  before  and  the  fly's  wing  as  object.  Focus 
and  light  well,  and  observe  carefully  the  appearance.  Rub  glycerin 
on  one  side  of  a  slide  near  the  end.  Hold  the  clean  side  of  this  end 
close  against  the  objective.  The  image  will  be  obscured,  and  cannot 
be  made  clear  by  focusing.  Then  use  a  clean  slide  and  the  image  may 
be  made  clear  by  elevating  the  tube  slightly.  The  obscurity  produced 
in  this  way  is  like  that  caused  by  clouding  the  front-lens  of  the  objec- 
tive. Dust  would  make  a  dark  patch  on  the  image  that  would  remain 
stationary  while  the  object  or  ocular  is  moved. 

If  a  small  diaphragm  is  employed  and  it  is  close  to  the  object, 
only  the  central  part  of  the  field  will  be  illuminated,  and  around  the 
small  light  circle  will  be  seen  a  dark  ring  (Fig.  42).  If  the  diaphragm 
is  lowered  or  a  sufficiently  large  one  employed  the  entire  field  will  be 
lighted. 

§  132.  Relative  Position  of  Objects  or  parts  of  the  same 
object.  The  general  rule  is  that  objects  highest  up  come  into  focus 
last  in  focusing  up,y?r5/  in  focusing  down. 

§  133.  Objects  having  Plane  or  Irregular  Outlines. — As  object 
use  three  printed  letters  in  stairs  mounted  in  Canada  balsam  (Fig.  89). 
The  first  letter  is  placed  directly  upon  the  slide,  and  covered  with  a 


CH.  Ill]  INTERPRETA  TION  OF  APPEARANCES  93 

small  piece  of  glass  about  as  thick  as  a  slide.  The  second  letter  is 
placed  upon  this  and  covered  in  like  manner.  The  third  letter  is  placed 
upon  the  second  thick  cover  and  covered  with  an  ordinary  cover- glass. 
The  letters  should  be  as  near  together  as  possible,  but  not  over-lapping. 
Employ  the  same  ocular  and  objective  as  above  (§  130). 


•  FIG.  89.   Letters  mounted  in  stairs  to 

show  the  order  of  coming  into  focus. 

d          _ 

;    \  A    g. .  a,  6,  c,  d.     The  various  letters  indi- 


cated  by  the  oblique  row  of  black  marks  in 
sectional  view.     Slide.     The  glass  slide  on  which  the  letters  are  mounted. 

Lower  the  tube  till  the  objective  almost  touches  the  top  letter,  then 
look  into  the  microscope,  and  slowly  focus  up.  The  lowest  letter  will 
first  appear,  and  then,  as  it  disappears,  the  middle  one  will  appear,  and 
so  on.  Focus  down,  and  the  top  letter  will  first  appear,  then  the  mid- 
dle one,  etc.  The  relative  position  of  objects  is  determined  exactly  in 
this  way  in  practical  work. 

For  example,  if  one  has  a  micrometer  ruled  on  a  cover-glass  15-25 
hundredths  mm.  thick,  it  is  not  easy  to  determine  with  the  naked  eye 
which  is  the  ruled  surface.  But  if  one  puts  the  micrometer  under  a 
microscope  and  uses  a  3  mm.(*^  in.)  objective,  it  is  easily  determined. 
The  cover  should  be  laid  on  a  slide  and  focused  till  the  lines  are  sharp. 
Now,  without  changing  the  focus  in  the  least  turn  the  cover  over.  If 
it  is  necessary  to  focus  up  to  get  the  lines  of  the  micrometer  sharp,  the 
lines  are  on  the  upper  side.  If  one  must  focus  down,  the  lines  are  on 
the  under  surface.  With  a  thin  cover  and  delicate  lines  this  method  of 
determining  the  position  of  the  rulings  is  of  considerable  practical 
importance. 

§  134.  Determination  of  the  Form  of  Objects. — The  procedure 
is  exactly  as  for  the  determination  of  the  form  of  large  objects.  That 
is,  one  must  examine  the  various  aspects.  For  example,  if  one  were 
placed  in  front  of  a  wall  of  some  kind  he  could  not  tell  whether  it  was 
a  simple  wall  or  whether  it  was  one  side  of  a  building  unless  in  some 
way  he  could  see  more  than  the  face  of  the  wall.  In  other  words,  in 
order  to  get  a  correct  notion  of  any  body,  one  must  examine  more  than 
one  dimension, — two  for  plane  surfaces,  three  for  solids.  So  for  micro- 
scopic objects,  one  must  in  some  way  examine  more  than  one  face.  To 
do  this  with  small  bodies  in  a  liquid  the  bodies  may  be  made  to  roll 
over  by  pressing  on  one  edge  of  the  cover-glass.  And  in  rolling  over 
the  various  aspects  are  presented  to  the  observer.  With  solid  bodies, 


94 


INTERPRETATION  OF  APPEARANCES 


\_CH.  Ill 


like  the  various  organs,  correct  notions  of  the  form  of  the  elements  can 
be  determined  by  studying  sections  cut  at  right  angles  to  each  other. 
The  methods  of  getting  the  elements  to  roll  over,  and  of  sectioning  in 
different  planes  are  in  constant  use  in  Histology,  and  the  microscopist 
who  neglects  to  see  all  sides  of  the  tissue  elements  has  a  very  inade- 
quate and  often  a  very  erroneous  conception  of  their  true  form. 

§  135.  Transparent  Objects  having  Curved  Outlines.  —  The 
success  of  these  experiments  will  depend  entirely  upon  the  care  and  skill 
used  in  preparing  the  objects,  in  lighting,  and  in  focusing. 

Employ  a  3  mm.  (^  in.)  or  higher  objective  and  a  high  ocular  for 
all  the  experiments.  It  may  be  necessary  to  shade  the  object  (§  109) 
to  get  satisfactory  results.  When  a  diaphragm  is  used  the  opening 
should  be  small  and  it  should  be  close  to  the  object. 

§  136.  Air  Bubbles.  —  Prepare  these  by  placing  a  drop  of  thin 
mucilage  on  the  center  of  a  slide  and  beating  it  with  a  scalpel  blade 
until  the  mucilage  looks  milky  from  the  inclusion  of  air  bubbles.  Put 
on  a  cover-glass  but  do  not  press  it  down. 


FIG.  90.  Diagram  show- 
ing  how  to  place  a'  cover- 
glass  upon  an  object  with  the 
forceps. 


§  137.  Air  Bubbles  with  Central  Illumination.  —  Shade  the 
object  ;  and  with  the  plane  mirror,  light  the  field  with  central  light 
(Fig.  23). 

Search  the  preparation  until  an  air  bubble  is  found  appearing 
about  i  mm.  in  diameter,  get  it  into  the  center  of  the  field,  and  if  the 
light  is  central  the  air  bubble  will  appear  with  a  wide,  dark,  circular 
margin  and  a  small  bright  center.  If  the  bright  spot  is  not  in  the 
center,  adjust  the  mirror  until  it  is. 

This  is  one  of  the  simplest  and  surest  methods  of  telling  when  the 
light  is  central  or  axial  when  no  condenser  is  used  (§  65). 

Focus  both  up  and  down,  noting  that,  in  focusing  up,  the  central 
spot  becomes  very  clear  and  the  black  ring  very  sharp.  On  elevating 
the  tube  of  the  microscope  still  more  the  center  becomes  dim,  and  the 
whole  bubble  loses  its  sharpness  of  outline. 

§  138.  Air  Bubbles  with  Oblique  Illumination.  —  Remove  the 
sub-stage  of  the  microscope  and  all  the  diaphragms.  Swing  the  mirror 
so  that  the  rays  may  be  sent  very  obliquely  upon  the  object  (Fig.  2^ 


CH.  Ill}  INTERPRE  TA  TION  OF  APPEARANCES  95 

C).  The  bright  spot  will  appear  no  longer  in  the  center  but  on  the 
side  away  from  the  mirror  (Fig.  91). 

§  r39-  Oil  Globules. — Prepare  these  by  beating  a  small  drop  of 
clove  oil  with  mucilage  on  a  slide  and  covering  as  directed  for  air  bub- 
bles (§  137),  or  use  a  drop  of  milk. 

§  140.  Oil  Globules  with  Central  Illumination. — Use  the  same 
diaphragm  and  light  as  above  (§137).  Find  an  oil  globule  appearing 
about  i  mm.  in  diameter.  If  the  light  is  central  a  bright  spot  will  ap- 
pear in  the  center  as  with  air.  Focus  up  and  down  as  with  air,  and 
note  that  the  bright  center  of  the  oil  globule  is  clearest  last  in  focus- 
ing up. 

FIG.  91.     Very  small  Globule  of  Oil  (O)  and  an   Air  Bubble  A 

(A)  seen  by  Oblique  Light.     The  arrow  indicates  the  direction  of 
the  light  rays. 

§  141.  Oil  Globules  with  Oblique  Illumination.— 
Remove  the  sub-stage,  etc.,  as  above,  and  swing  the  mir- 
ror to  one  side  and  light  with  oblique  light.  The  bright 
spot  will  be  eccentric,  and  will  appear  to  be  on  the  same 
side  as  the  mirror  (Fig.  91). 

§  142.  Oil  and  Air  Together. — Make  a  prepara- 
tion exactly  as  described  for  air  bubbles  (§  136),  and  add 
at  one  edge  a  little  of  the  mixture  of  oil  and  mucilage 
(§  T39)  ;  cover  and  examine. 

The  sub-stage  need  not  be  used  in  this  experiment.  Search  the 
preparation  until  an  air  bubble  and  an  oil  globule,  each  appearing 
about  i  mm.  in  diameter,  are  found  in  the  same  field  of  view.  Light 
first  with  central  light,  and  note  that,  in  focusing  up,  the  air  bubble 
comes  into  focus  first  and  that  the  central  spot  is  smaller  than  that  of 
the  oil  globule.  Then,  of  course,  the  black  ring  will  be  wider  in  the 
air  bubble  than  in  the  oil  globule.  Make  the  light  oblique.  The 
bright  spot  in  the  air  bubble  will  move  away  from  the  mirror  while 
that  in  the  oil  globule  will  move  toward  it.  See  Fig.  91.* 

§  143.  Air  and  Oil  by  Reflected  Light. — Cover  the  diaphragm 
or  mirror  so  that  no  transmitted  light  (§  64)  can  reach  the  preparation, 
using  the  same  preparation  as  in  §  142.  The  oil  and  air  will  appear 
like  globules  of  silver  on  a  dark  ground.  The  part  that  was  darkest  in 


*It  should  be  remembered  that  the  image  in  the  compound  microscope  is 
inverted  (Fig.  21),  hence  the  bright  spot  really  moves  toward  the  mirror  for  air, 
and  away  from  it  for  oil. 


96  INTERPRETATION  OF  APPEARANCES  [C7/.  Ill 

each  will  be  lightest,  and  the  bright  central  spot  will  be  somewhat 
dark.* 

§  144.  Distinctness  of  Outline. — In  refraction  images  this 
depends  on  the  difference  between  the  refractive  power  of  a  body  and 
that  of  the  medium  which  surrounds  it.  The  oil  and  air  were  very 
distinct  in  outline  as  both  differ  greatly  in  refractive  power  from  the 
medium  which  surrounds  them,  the  oil  being  more  refractive  than  the 
mucilage  and  the  air  less.  (Figs.  54-56.) 

Place  a  fragment  of  a  cover-glass  on  a  clean  slide,  and  cover  it 
(see  under  mounting).  The  outline  will  be  distinct  with  the  unaided 
eye.  Use  it  as  object  and  employ  the  16  mm.  (^i  in.)  objective  and 
high  ocular.  Light  with  central  light.  The  fragment  will  be  outlined 
by  a  dark  band.  Put  a  drop  of  water  at  the  edge  of  the  cover-glass. 
It  will  run  in  and  immerse  the  fragment.  The  outline  will  remain  dis- 
tinct, but  the  dark  band  will  be  somewhat  narrower.  Remove  the 
cover-glass,  wipe  it  dry,  and  wipe  the  fragment  and  slide  dry  also. 
Put  a  drop  of  50%  glycerin  on  the  middle  of  the  slide  and  mount  the 
fragment  of  cover-glass  in  that.  The  dark  contour  will  be  much  nar- 
rower than  before. 

Draw  a  solid  glass  rod  out  to  a  fine  thread.  Mount  one  piece  in 
air,  and  the  other  in  50%  glycerin.  Put  a  cover-glass  on  each.  Em- 
ploy the  same  optical  arrangement  as  before.  Examine  the  one  in  air 
first.  There  will  be  seen  a  narrow,  bright  band,  with  a  wide,  dark 
band  on  each  side. 

The  one  in  glycerin  will  show  a  much  wider  bright  central  band, 
with  the  dark  borders  correspondingly  narrow  (Fig.  92,  b).  The  dark 
contour  depends  also  on  the  numerical  aperture  of  the  objective — being 
wider  with  low  apertures.  This  can  be  readily  understood  when  it  is 
remembered  that  the  greater  the  aperture  the  more  oblique  the  rays  of 
light  that  can  be  received,  and  the  dark  band  simply  represents  an 
area  in  which  the  rays  are  so  greatly  bent  or  refracted  (Figs.  54-56) 
that  they  cannot  enter  the  objective  and  contribute  to  the  formation  of 
the  image  ;  the  edges  are  dark  simply  because  no  light  from  them 
reaches  the  observer. 


*It  is  possible  to  distinguish  oil  and  air  optically,  as  described  above,  only 
when  quite  high  powers  are  used  and  very  small  bubbles  are  selected  for  observa- 
tion. If  a  1 6  mm.  (%  in.)  is  used  instead  of  a  3  mm.  (ft  in.)  objective,  the  ap- 
pearances will  vary  considerably  from  that  given  above  for  the  higher  power.  It 
is  well  to  use  a  low  as  well  as  a  high  power.  Marked  differences  will  also  be 
seen  in  the  appearances  with  objectives  of  small  and  of  large  aperture. 


CH.  Ill  ]  INTERPRE  TA  TION  OF  APPEARANCES  97 

FIG.  92.  Solid  glass  rod  showing  the 
appearance  when  viewed  with  transmit- 
ted, central  light,  and  with  an  objective 
of  medium  aperture. 

a.  Mounted  in  air.     b.  Mounted  in  50 per  cent,  glycerin. 

If  the  glass  rod  or  any  other  object  were  mounted  in  a  medium  of 
the  same  color  and  refractive  power,  it  could  not  be  distinguished  from 
the  medium.* 

A  very  striking  and  satisfactory  demonstration  may  be  made  by 
painting  a  zone  or  band  of  eosin  or  other  transparent  color  on  a  solid 
glass  rod,  and  immersing  the  rod  in  a  test  tube  or  vial  of  cedar  oil; 
clove  oil  or  turpentine.  Above  the  liquid  the  glass  rod  is  very  evident, 
as  it  is  also  at  the  colored  zone,  but  at  other  levels  it  can  hardly  be 
seen  in  the  liquid. 

§  145.  Highly  Refractive. — This  expression  is  often  used  in  de- 
scribing microscopic  objects,  (medullated  nerve  fibers,  for  example), 
and  means  that  the  object  will  appear  to  be  bordered  by  a  wide,  dark 
margin  when  it  is  viewed  by  transmitted  light.  And  from  the  above 
(§  144),  it  would  be  known  that  the  refractive  power  of  the  object,  and 
the  medium  in  which  it  was  mounted  must  differ  considerably. 

§  146.  Doubly  Contoured. — This  means  that  the  object  is 
bounded  by  two,  usually  parallel  dark  lines  with  a  lighter  band  between 
them.  In  other  words,  the  object  is  bordered  by  (i)  a  dark  line, (2)  a 
light  band,  and  (3)  a  second  dark  line  (Fig.  93). 

This  may  be  demonstrated  by  coating  a  fine  glass  rod  (§  144)  with 
one  or  more  coats  of  collodion  or  celloidin  and  allowing  it  to  dry,  and 
then  mounting  in  50%  glycerin  as  above.  Employ  a  3  mm.  (^6  in.)  or 
higher  objective,  light  with  transmitted  light,  and  it  will  be  seen  that 
where  the  glycerin  touches  the  collodion  coating  there  is  a  dark  line — 
next  this  is  a  light  band,  and  finally  there  is  a  second  dark  line  where 
the  collodion  is  in  contact  with  the 
glass  rod.*  (Fig.  93). 

FIG.  93.     Solid  glass  rod  coated  with  col- 1 
lodion  to  show  a  double  contour.     Toward  I 
one  end  the  collodion  had  gathered  in  afusi-    \ 
form  drop. 


*Some  of  the  rods  have  air  bubbles  in  them,  and  then  there  results  a  capillary 
tube  when  they  are  drawn  out.  It  is  well  to  draw  out  a  glass  tube  into  a  fine 
thread  and  examine  it  as  described.  The  central  cavity  makes  the  experiment 
much  more  complex. 


98  INTERPRETATION  OF  APPEARANCES  \_CH.  HI 

§  147.  Optical  Section. — This  is  the  appearance  obtained  in 
examining  transparent  or  nearly  transparent  objects  with  a  microscope 
when  some  plane  below  the  upper  surface  of  the  object  is  in  focus. 
The  upper  part  of  the  object  which  is  out  of  focus  obscures  the  image 
but  slightly.  By  changing  the  position  of  the  objective  or  object,  a 
different  plane  will  be  in  focus  and  a  different  optical  section  obtained. 
The  most  satisfactory  optical  sections  are  obtained  with  high  objectives 
having  large  aperture. 

Nearly  all  the  transparent  objects  studied  may  be  viewed  in  optical 
section.  A  striking  example  will  be  found  in  studying  mammalian 
red  blood-corpuscles  on  edge.  The  experiments  with  the  solid  glass 
rods  (Fig.  92)  furnish  excellent  and  striking  examples  of  optical 
sections. 

§  148.  Currents  in  Liquids. — Employ  the  16  mm.  (^  in.)  ob- 
jective, and  as  object  put  a  few  particles  of  carmine  on  the  middle  of  a 
slide,  and  add  a  drop  of  water.  Grind  the  carmine  well  with  a  scalpel 
blade,  and  then  cover  it.  If  the  microscope  is  inclined,  a  current  will 
be  produced  in  the  water,  and  the  particles  of  carmine  will  be  carried 
along  by  it.  Note  that  the  particles  seem  to  flow  up  instead  of  down — 
why  is  this  ? 

Lamp-black  rubbed  in  water  containing  a  little  mucilage  answers 
well  for  this  experiment. 

§  149.  Velocity  Under  the  Microscope. — In  studying  currents 
or  the  movement  of  living  things  under  the  microscope,  one  should 
not  forget  that  the  apparent  velocity  is  as  unlike  the  real  velocity  as 
the  apparent  size  is  unlike  the  real  size.  If  one  consults  Fig.  37  it 
will  be  seen  that  the  actual  size  of  the  field  of  the  microscope  with  the 
different  objectives  and  oculars  is  inversely  as  the  magnification.  That 
is,  with  great  magnification  only  a  small  area  can  be  seen.  The  field 
appears  to  be  large,  however,  and  if  any  object  moves  across  the  field 
it  may  appear  to  move  with  great  rapidity,  whereas  if  one  measures 
the  actual  distance  passed  and  notes  the  time,  it  will  be  seen  that  the 
actual  motion  is  quite  slow.  One  should  keep  this  in  mind  in  study- 
ing the  circulation  of  the  blood.  The  truth  of  what  has  just  been 
said  can  be  easily  demonstrated  in  studying  the  circulation  in  the  gills 

*The  collodion  used  is  a  6%  solution  of  gun  cotton  in  equal  parts  of  sulphuric 
ether  and  95%  alcohol.  It  is  well  to  dip  the  rod  two  or  three  times  in  the  collo- 
dion and  to  hold  it  vertically  while  drying.  The  collodion  will  gather  in  drops, 
and  one  will  see  the  difference  between  a  thick  and  a  thin  membranous  covering. 
(Fig.  93). 


CH.  Ill}  INTERPRETATION  OF  APPEARANCES  99 

of  Necturus,  or  in  the  frog's  foot,  by  using  first  a  low  power  in  which 
the  field  is  actually  of  considerable  diameter  (Fig.  37,  Table,  §  51)  and 
then  using  a  high  power.  With  the  high  power  the  apparent  motion 
will  appear  much  more  rapid.  For  spiral,  serpentine  and  other  forms 
of  motion,  see  Carpenter-Dallinger,  p.  433. 

§  150.  Pedesis  or  Brownian  Movement. — Employ  the  same 
object  as  above,  but  a  3  mm.  (^i  in.)  or  higher  objective  in  place  of 
the  1 6  mm.  Make  the  body  of  the  microscope  vertical,  so  that  there 
may  be  no  currents  produced.  Use  a  small  diaphragm  and  light  the 
field  well.  Focus  and  there  will  be  seen  in  the  field  large  motionless 
masses,  and  between  them  small  masses  in  constant  motion.  This  is 
an  indefinite,  dancing  or  oscillating  motion. 

This  indefinite  but  continuous  motion  of  small  particles  in  a  liquid 
is  called  Pe-de'sis  or  Brownian  movement.  Also,  but  improperly,  molec- 
ular movement,  from  the  smallness  of  the  particles. 

The  motion  is  increased  by  adding  a  little  gum  arabic  solution  or 
a  slight  amount  of  silicate  of  soda  or  soap  ;  sulphuric  acid  and  various 
saline  compounds  retard  or  check  the  motion.  One  of  the  best  objects 
is  lamp-black  ground  up  with  a  little  gum  arabic.  Carmine  prepared 
in  the  same  way,  or  simply  in  water,  is  excellent  ;  and  very  finely 
powdered  pumice-stone  in  water  has  for  many  years  been  a  favorite 
object. 

Pedesis  is  exhibited  by  all  solid  matter  if  it  is  finely  enough  di- 
vided and  in  a  suitable  liquid.  In  the  minds  of  most,  no  adequate 
explanation  has  yet  been  offered.  See  Carpenter-Dallinger,  p.  431  ; 
Beale,  p.  195  ;  Jevons  in  Quart.  Jour.  Science,  n.  s.,  Vol.  VIII  (1878), 
p.  167.  In  1894,  Meade  Bache  published  a  paper  in  the  Proc.  Amer. 
Philos.  Soc.,  Vol.  XXXIII,  pp.  163-167,  entitled  "The  Secret  of 
the  Brownian  Movement."  This  paper  is  suggestive  if  not  very 
satisfactory. 

For  the  orginal  account  of  this  see  Robert  Brown,  "Botanical 
appendix  to  Captain  King's  voyage  to  Australia,"  Vol.  II,  p.  534. 
(1826). 

See  also  Dr.  C.  Aug.  Sigm.  Schultze,  "Mikroskopische  Unter- 
suchungen  u'ber  des  Herren  Robert  Brown  Entdeckunglebender,  selbst 
im  Feuer  unzerstorbarer  Theilchen  in  alien  Korpern."  From  "Die 
Gesellschaft  fur  Belorderung  der  Naturwissenschaften  zu  Freiburg. ' ' 
1828. 

Compare  the  pedetic  motion  with  that  of  a  current  by  slightly  in- 
clining the  tube  of  the  microscope.  The  small  particles  will  continue 


ioo  INTERPRE TA  TION  OF  APPEARANCES  .          [CH.  Ill 

their  independent  leaping  movements  while  they  are  carried  along  by 
the  current.  The  pedetic  motion  makes  it  difficult  to  obtain  good 
photographs  of  milk  globules  and  other  small  particles.  The  difficulty 
may  be  overcome  by  mixing  the  milk  with  a  very  weak  solution  of 
gelatin  and  allowing  it  to  cool  (see  Ch.  .IX). 

§  151.  Demonstration  of  Pedesis  with  the  Polarizing  Micro- 
scope (Ch.  VI). — The  following  demonstration  shows  conclusively 
that  the  pedetic  motion  is  real  and  not  illusive.  (Ranvier,  p.  173.) 

Open  the  abdomen  of  a  dead  frog  (an  alcoholic  or  formalin 
specimen  is  satisfactory).  Turn  the  viscera  to  one  side  and  observe 
the  small,  whitish  masses  at  the  emergence  of  the  spinal  nerves.  With 
fine  forceps  remove  one  of  these  and  place  it  on  the  middle  of  a  clean 
slide.  Add  a  drop  of  water,  or  of  water  containing  a  little  gum  arabic. 
Rub  the  white  mass  around  in  the  drop  of  liquid  and  soon  the  liquid 
will  have  a  milky  appearance.  Remove  the  white  mass,  place  a  cover- 
glass  on  the  milky  liquid  and  seal  the  cover  by  painting  a  ring  of 
castor  oil  all  around  it,  half  the  ring  being  on  the  slide  and  half 
on  the  cover-glass.  This  is  to  avoid  the  production  of  currents  by 
evaporation. 

Put  the  preparation  under  the  microscope  and  examine  with,  first 
a  low  then  a  high  power  (3  mm.  or  ^  in.).  In  the  field  wilt  be  seen 
multitudes  of  crystals  of  carbonate  of  lime  ;  the  larger  crystals  are 
motionless  but  the  smallest  ones  exhibit  marked  pedetic  movement. 

Use  the  micro-polariscope,  light  with  great  care  and  exclude  all 
adventitious  light  from  the  microscope  by  shading  the  object  (§  109)  and 
also  by  shading  the  eye.  Focus  sharply  and  observe  the  pedetic  motion 
of  the  small  particles,  then  cross  the  polarizer  and  analyzer,  that  is, 
turn  one  or  the  other  until  the  field  is  dark.  Part  of  the  large  motion- 
less crystals  will  shine  continuously  and  a  part  will  remain  dark,  but 
small  crystals  between  the  large  ones  will  shine  for  an  instant,  then 
disappear,  only  to  appear  again  the  next  instant.  This  demonstration 
is  believed  to  furnish  absolute  proof  that  the  pedetic  movement  is  real 
and  not  illusory. 

§  152.  Muscae  Volitantes. — These  specks  or  filaments  in  the 
eyes  due  to  minute  shreds  or  opacities  of  the  vitreous  sometimes  appear 
as  part  of  the  object  as  they  are  projected  into  the  field  of  vision.  They 
may  be  seen  by  looking  into  the  well  lighted  microscope  when  there  is 
no  object  under  the  microscope.  They  may  also  be  seen  by  looking 
at  brightly  illuminated  snow  or  other  white  surface.  By  studying 
them  carefully  it  will  be  seen  that  they  are  somewhat  movable  and  float 


CH.  III  INTERPRETA  TION  OF  APPEARANCES  101 


across  the  field  of  vision,  and  thus  do  not  remain  in  one  position  as  do 
the  objects  under  observation.  Furthermore,  one  may,  by  taking  a 
little  pains,  familiarize  himself  with  the  special  forms  in  his  own  eyes 
so  that  the  more  conspicuous  at  least  may  be  instantly  recognized. 

§  153.  In  addition  to  the  above  experiments  it  is  very  strongly 
recommended  that  the  student  follow  the  advice  of  Beale,  p.  248,  and 
examine  first  with  a  low  then  with  a  higher  power,  mounted  dry,.  then 
in  water,  lighted  with  reflected  light,  then  with  transmitted  light,  the 
following  :  Potato,  wheat,  rice,  and  corn  starch,  easily  obtained  by 
scraping  the  potato  and  the  grains  mentioned  ;  bread  crumbs  ;  portions 
of  feather.  Portions  of  feather  accidentally  present  in  histological 
preparations  have  been  mistaken  for  lymphatic  vessels  (Beale,  288). 
Fibers  of  cotton,  linen  and  silk.  Textile  fibers  accidentally  present 
have  been  considered  nerve  fibers,  etc.  Human  and  animal  hairs. 
Study  with  especial  care  hairs  from  various  parts  of  the  body  of  the 
animals  used  for  dissection  in  the  laboratory  where  you  work.  These 
are  liable  to  be  present  in  histological  preparations,  and  unless  their 
character  is  understood  there  is  chance  for  much  confusion  and  erro- 
neous interpretation.  The  scales  of  butterflies  and  moths,  especially 
the  common  clothes  moth.  The  dust  swept  from  carpeted  and  wood 
floors.  Tea  leaves  and  coffee  grounds.  Dust  found  in  living  rooms 
and  places  not  frequently  dusted.  In  the  last  will  be  found  a  regular 
museum  of  objects. 

For  figures  (photo-micrographs,  etc.)  of  the  various  forms  of  starch, 
see  Bulletin  No.  13  of  the  Chemical  Division  of  the  U.  S.  Department 
af  Agriculture.  For  Hair  and  Wool,  see  Bulletin  of  the  National  Asso- 
ciation of  Wool  Growers,  1875,  P-  47°>  Proc.  Amer.  Micr.  Soc.,  1884, 
pp.  65-68.  Herzfeld,  translated  by  Salter.  —  The  technical  testing  of 
yarns  and  textile  fabrics,  London,  1898. 

For  different  appearances  due  to  the  illuminator,  see  Nelson,  in 
Jour.  Roy,  Micr.  Soc.,  1891,  pp.  90-105  ;  and  for  the  illusory  appear- 
ances due  to  diffraction  phenomena,  see  Carpenter-Dallinger,  p.  434. 
Mercer.  Trans.  Amer.  Micr.  Soc.,  pp.  321-396. 

If  it  is  necessary  to  see  all  sides  of  an  ordinary  gross  object,  and 
to  observe  it  with  varying  illumination  and  under  various  conditions  of 
temperature,  moisture,  etc.,  in  order  to  obtain  a  fairly  accurate  and 
satisfactory  knowledge  of  it,  so  much  the  more  is  it  necessary  not  to  be 
satisfied  in  microscopical  observation  until  every  means  of  investigation 
and  verification  has  been  called  into  service,  and  then  of  the  image 
that  falls  upon  the  retina,  only  such  details  will  be  noted  as  the  brain 
behind  the  eye  is  ready  to  appreciate. 


102 


INTERPRETATION  OF  APPEARANCES 


[CH.  Ill 


To  summarize  this  chapter  and  leave  with  the  beginning  student 
the  result  of  the  experience  of  many  eminent  workers  : 

1.  Get  all  the  information  possible  with  the  unaided  eye.     See 
the  whole  object  and  all  sides  of  it,  so  far  as  possible. 

2.  Examine  the  preparation  with  a  simple  microscope  in  the  same 
thorough  way  for  additional  detail. 

3.  Use  a  low  power  of  the  compound  microscope. 

4.  Use  a  higher  power. 

5.  Use  the  highest  power  available  and  applicable.     In  this  way 
one  sees  the  object  as  a  whole  and  progressively  more  and  more  details. 
Then  as  the  object  is  viewed  from  two  or  more  aspects,  something  like 
a  correct  notion  may  be  gained  of  its  form  and  structure. 


THE  MICROSCOPE  IN  SECTION 


1.  Positive  ocular. 

2.  Draw-tube. 

3.  Main  tube  or  body. 

4-5.  Society    screws    in    the 
^draw-tube  and   body. 

6.  Objective  in  position. 

7.  Stage. 

8.  Spring  for  holding 

slides. 

9.  Sub-stage  condenser. 
10.  Iris  diaphragm. 


11.  Plane  and  concave  mir- 

ror. 

12.  Horse-shoe  base. 

13.  Rack    and    pinion    for 

condenser. 

14.  Flexible  pillar. 

15.  Spiral  spring  of  fine  ad- 

justment. 

16.  Fine  adjustment 

17.  Coarse  adjustment. 


CHAPTER  IV 


MAGNIFICATION   AND    MICROMETRY 


APPARATUS   AND   MATERIAL    FOR    THIS    CHAPTER 

Simple  and  compound  microscope  (§  156,  158);  Steel  scale  or  rule  divided  to 
millimeters  and  iths  ;  Block  for  magnifier  and  compound  microscope  (§  156,  160); 
Dividers  (§  156,  160);  Stage  micrometer  (g  159);  Wollaston  camera  lucida  (§  160); 
Ocular  screw-micrometers  (Figs.  106-107);  Micrometer  ocular  (Figs.  104-105). 
Abbe  camera  lucida  (Fig.  101).  Necturus  red  blood  corpuscles  (§  168). 

§  154.  The  Magnification,  Amplification  or  Magnifying  Power 
of  a  simple  or  compound  microscope  is  the  ratio  between  the  real  and 
the  apparent  size  of  the  object  examined.  The  apparent  size  is  ob- 
tained by  measuring  the  virtual  image  (Figs.  21,  38).  The  object  for 
determining  magnification  must  be  of  known  length  and  is  designated 
a  micrometer  (§  159).  In  practice  a  virtual  image  is  measured  by  the 
aid  of  some  form  of  camera  lucida  (Figs.  97,  101),  or  by  double  vision 
(§156).  As  the  length  of  the  object  is  known,  the  magnification  is 
easily  determined  by  dividing  the  apparent  size  of  the  image  by  the 
actual  size  of  the  object.  For  example,  if  .the  virtual  image  measures 
40  mm.  and  the  object  magnified,  2  mm.,  the  amplification  must  be 
40  -r-  2  =  20,  that  is,  the  apparent  size  is  20  fold  greater  than  the  real 
size. 

Magnification  is  expressed  in  diameters  or  times  linear,  that  is,  but 
one  dimension  is  considered.  In  giving  the  scale  at  which  a  micro- 
scopical or  histological  drawing  is  made,  the  word  magnification  is  fre- 
quently indicated  by  the  sign  of  multiplication  thus  :  X  450,  upon  a 
drawing  would  mean  that  the  figure  or  drawing  is  450  times  as  large 
as  the  object. 

§  155.  Magnification  of  Real  Images.— In  this  case  the  mag- 
nification is  the  ratio  between  the  size  of  the  real  image  and  the  size  of 
the  object,  and  the  size  of  the  real  image  can  be  measured  directly.  By 
recalling  the  work  on  the  function  of  an  objective  (§  53),  it  will  be 
remembered  that  it  forms  a  real  image  on  the  ground  glass  placed  on 
the  top  of  the  tube,  and  that  this  real  image  could  be  looked  at  with  the 


104 


MAGNIFICATION  AND  MICROMETRY 


[C//.  IV 


eye  or  measured  as  if  it  were  an  actual  object.  For  example,  suppose 
the  object  were  three  millimeters  long  and  its  image  on  the  ground  glass 
measured  15  mm.,  then  the  magnification  must  be,  15  -r-  3  =  5,  that  is, 
the  real  image  is  5  times  as  long  as  the  object.  The  real  images  seen 
in  photography  are  mostly  smaller  than  the  objects,  but  the  magnifica- 
tion is  designated  in  the  same  way  by  dividing  the  size  of  the  real  im- 
age measured  on  the  ground  glass  by  the  size  of  the  object.  For 
example,  if  the  object  is  400  millimeters  long  and  its  image  on  the 
ground  glass  is  '25  mm.  long,  the  ratio  is  25^-400=-^.  That  is,  the 
image  is  ^g-th  as  long  as  the  object  and  is  not  magnified  but  reduced. 
In  marking  negatives,  as  with  drawings,  the  sign  of  multiplication  is 
put  before  the  ratio,  and  in  the  example  the  designation  would  be 
XTVth. 

MAGNIFICATION    OF   A   SIMPLE    MICROSCOPE 

§  156.  The  Magnification  of 
a  Simple  Microscope  is  the  ratio 
between  the  object  magnified  (Fig. 
1 6,  A'B'),  and  the  virtual  image 
(A3B3).  To  obtain  the  size  of  this 
virtual  image  place  the  tripod  mag- 
nifier near  the  edge  of  a  support  of 
such  a  height  that  the  distance 
from  the  upper  surface  of  the  mag- 
nifier to  the  table  is  250  millimeters. 

FIG.  94.      Tripod  Magnifier. 

As  object,  place  a  scale  of  some  kind  ruled  in  millimeters  on  the 
support  under  the  magnifier.  Put  some  white  paper  on  the  table  at 
the  base  of  the  support  and  on  the  side  facing  the  light. 


0 


FIG.  95.     Ten  Centimeter  Rule.     The  upper  edge  is  divided  into  millimeters, 
the  lower  into  centimeters  at  the  left  and  half  centimeters  at  the  right. 

Close  one  eye,  and  hold  the  head  so  that  the  other  will  be  near  the 
upper  surface  of  the  lens.     Focus  if  necessary  to  make  the  image  clear 


CH.  1 V  ]  MA  GNIFICA  TION  A  ND  MICRO  ME  TRY  105 

(§  n).  Open  the  closed  eye  and  the  image  of  the  rule  will  appear  as 
if  on  the  paper  at  the  base  of  the  support.  Hold  the  head  very  still, 
and  with  dividers  get  the  distance  between  any  two  lines  of  the  image. 
This  is  the  so-called  method  of  double  vision  in  which  the  microscope 
image  is  seen  with  one  eye  and  the  dividers  with  the  other,  the  two 
images  appearing  to  be  fused  in  a  single  visual  field. 

§  157.  Measuring  the  Spread  of  Dividers. — This  should  be 
done  on  a  steel  scale  divided  to  millimeters  and  -J-ths. 

As  \  mm.  cannot  be  seen  plainly  by  the  unaided  eye,  place  one 
arm  of  the  dividers  at  a  centimeter  line,  and  with  the  tripod  magnifier 
count  the  number  of  spaces  on  the  rule  included  between  the  points  of 
the  dividers.  The  magnifier  simply  makes  it  easy  to  count  the  spaces 
on  the  rule  included  between  the  points  of  the  dividers — it  does  not,  of 
course,  increase  the  number  of  spaces  or  change  their  value. 

As  the  distance  between  any  two  lines  of  the  image  of  the  scale 
gives  the  size  of  the  virtual  image  (Fig.  16,  A3B3),  and  as  the  size  of 
the  object  is  known,  the  magnification  is  determined  by  dividing  the 
size  of  the  image  by  the  size  of  the  object.  Thus,  suppose  the  distance 
between  the  two  lines  of  the  image  is  measured  by  the  dividers  and 
found  on  the  steel  scale  to  be  15  millimeters,  and  the  actual  size  of  the 
space  between  the  two  lines  of  the  object  is  2  millimeters,  then  the 
magnification  must  be  15^-2=7^.  That  is,  the  image  is  7^  times  as 
long  or  wide  as  the  object.  In  this  case  the  image  is  said  to  be 
magnified  7^  diameters,  or  7^2  times  linear. 

The  magnification  of  any  simple  magnifier  may  be  determined 
experimentally  in  the  wray  described  for  the  tripod. 

MAGNIFICATION    OF    A    COMPOUND    MICROSCOPE 

§  158.  The  Magnification  of  a  Compound  Microscope  is  the 
ratio  between  the  final  or  virtual  image  (Fig.  21,  B3A3),  and  the  object 
magnified  (A  B). 

The  determination  of  the  magnification  of  a  compound  microscope 
may  be  made  as  with  a  simple  microscope  (§  156),  but  this  is  very 
fatiguing  and  unsatisfactory. 

§  159.  Stage,  Object  or  Objective  Micrometer. — For  deter- 
mining the  magnification  of  a  compound  microscope  and  for  the 
purpose  of  micrometry,  it  is  necessary  to  have  a  finely  divided  scale 
or  rule  on  glass  or  on  metal.  Such  a  finely  divided  scale  is  called  a 
micrometer,  and  for  ordinary  work  one  mounted  on  a  glass  slide 
(i  X  3  in,  25  x  76  mm.)  is  most  convenient. 


io6  MAGNIFICATION  AND  MICROMETRY  [Cff.  IV 

The  spaces  between  the  lines  should  be  y1^  and  y^  mm.  (or  if  in 
inches,  y^  and  T^TF  i*1-)  Micrometers  are  sometimes  ruled  on  the 
slide,  but  more  satisfactorily  on  a  cover-glass  of  known  thickness, 
preferably  o.  15-0. 18  mm.  The  covers  should  be  perfectly  clean  before 
the  ruling,  and  afterwards  simply  dusted  off  with  a  camel's  hair 
duster,  and  then  mounted,  lines  downward  over  a  shellac  or  other 
good  cell.  (See  Ch.  VII).  If  one  rubs  the  lines  the  edges  of  the 
furrow  made  by  the  diamond  are  liable  to  be  rounded  and  the  sharp- 
ness of  the  micrometer  is  lost.  If  the  lines  are  on  the  slide  and  un- 
covered one  cannot  use  the  micrometer  with  an  oil  immersion,  as  the 
oil  obliterates  the  lines.  Cleaning  the  slide  makes  the  lines  less  sharp 
as  stated.  If  the  lines  are  very  coarse,  it  is  an  advantage  to  fill  them 
with  plumbago.  This  may  be  done  either  with  some  very  fine  plum- 
bago on  the  end  of  a  so  ft  cork,  or  by  using  an  exceedingly  soft  lead 
pencil.  Lines  properly  filled  may  be  covered  with  balsam  and  a  cover- 
glass  as  in  ordinary  balsam  mounting  (Ch.  VII). 

§  1 60.  Determination  of  Magnification. — This  is  most  readily 
accomplished  by  the  use  of  some  form  of  camera  lucida  (Ch.  V),  that 
of  Wollaston  being  most  convenient  as  it  may  be  used  for  all  powers, 
and  the  determination  of  the  standard  distance  of  250  millimeters  at 
which  to  measure  the  images  is  very  readily  determined  (Fig.  97,  §  162). 

Employ  the  16  mm.  (^i  in.)  objective  and  a  37  mm.  (or  X  8  ocu- 
lar with  a  stage  micrometer  as  object.  For  this  power  the  T^  mm. 
spaces  of  the  micrometer  should  be  used  as  object.  Focus  sharply. 

FIG.  96.     Diagram  of  a    stage    micrometer, 
with  a  ring  on  the  lines  to  facilitate  finding  them. 

It  is  somewhat  difficult  to  find  the  mi- 
crometer lines.  To  avoid  this  it  is  well  to 
have  a  small  ring  enclosing  some  of  the 

micrometer  lines  (Fig.  96).  The  light  must  also  be  carefully  regu- 
lated. If  too  much  light  is  used,  i.  e.,  too  large  an  aperture,  the  lines 
will  be  drowned  in  the  light.  In  focusing  with  the  high  powers  be 
very  careful.  Remember  the  micrometers  are  expensive,  and  one  can- 
not afford  to  break  them.  As  suggested  in  §  74,  focus  on  the  edge  of 
the  cement  ring  enclosing  the  lines,  then  in  focusing  down  to  find 
the  lines,  move  the  preparation  very  slightly,  back  and  forth. 

After  the  lines  are  sharply  focused,  and  the  slide  clamped  in  posi- 
tion make  the  tube  of  the  microscope  horizontal,  by  bending  the  flexible 
pillar,  being  careful  not  to  bring  any  strain  upon  the  fine  adjustment 
(frontispiece). 


CH. 


MAGNIFICATION  AND  MICROMETRY 


107 


FIG.  97. 


FIG.  97.  Wollaston^s  Camera  Lu- 
cida,  showing  the  rays  from  the  micro- 
scope and  from  the  drawing  surface, 
also  the  position  of  the  pupil  of  the  eye. 

Axis,  Axis.  Axial  rays  from 
the  microscope  and  from  the  drawing 
surface  (Ch.  V). 

Camera  Lucida.  A  section  of  the 
quadrangular  prism  showing  the 
course  of  the  rays  in  the  prism  from 
the  microscope  to  the  eye.  As  the  rays 
are  twice  reflected,  they  have  the  same 
relation  on  entering  the  eye  that  they 
would  have  by  looking  directly  into  the 
ocular. 

A.  B.  The  lateral  rays  from  the 
microscope  and  their  projection  upon 
the  drawing  surface. 

C.  D  Rays  .from  the  drawing 
surface  to  ths  eye. 

A.  D.  A'  D' .  Overlapping  portions  of  the  two  fields,  where  both  the  micro- 
scopic image  and  the  drawing  surf  ace,  pencil,  etc.,  can  be  seen.  It  is  represented 
by  the  shaded  part  of  the  overlapping  circles  at  the  right. 

Ocular.     The  ocular  of  the  microscope. 

P.     The  drawing  pencil.     Its  point  is  shown  in  the  overlapping  fields. 

Put  a  Wollaston  camera  lucida  (Fig.  97  and  Ch.  V)  in  position, 
and  turn  the  ocular  around  if  necessary  so  that  the  broad  flat  surface 
may  face  directly  upward,  as  shown  in  Fig.  97.  Elevate  the  micro- 
scope by  putting  a  block  under  the  base,  so  that  the  perpendicular  dis- 
tance from  the  upper  surface  of  the  camera  lucida  to  the  table  is  250 
mm.  (§  162).  Place  some  white  paper  on  the  work-table  beneath  the 
camera  lucida. 

Close  one  eye,  and  hold  the  head  so  that  the  other  may  be  very 
close  to  the  camera  lucida.  Look  directly  down.  The  image  will  ap- 
pear to  be  on  the  table.  It  may  be  necessary  to  readjust  the  focus 
after  the  camera  lucida  is  in  position.  If  there  is  difficulty  in  seeing 
dividers  and  image  consult  Ch.  V.  Measure  the  image  with  dividers 
and  obtain  the  power  exactly  as  above  (§156-157). 

Thus  :  Suppose  two  of  the  yV^h  mm.,  spaces  were  taken  as  object, 
and  the  image  is  measured  by  the  dividers,  and  the  spread  of  the 
dividers  is  found  on  the  steel  rule  to  be  9-f  millimeters.  If  now  the 
object  is  y^ths  of  a  millimeter  and  the  magnified  image  is  9f  milli- 
meters, the  magnification  (which  is  the  ratio  between  size  of  object 


io8 


MAGNIFICATION  AND  MICROMETRY 


[CH.  IV 


and  image)  must  be  g|  -r-  fV  =  47.  That  is,  the  magnification  is  47 
diameters,  or  47  times  linear.  If  the  fractional  numbers  in  the  above 
example  trouble  the  student,  both  may  be  reduced  to  the  same  denom- 
ination, thus  :  If  the  size  of  the  image  is  found  to  be  9-f  mm.  this 
number  may  be  reduced  to  tenths  mm.,  so  it  will  be  of  the  same 

Image 


FIG.  99. 


FIGS,  98-99.  Figures  showing  that  the  size  of  object  and  image  very  directly 
as  their  distance  from  the  center  of  the  lens.  In  Fig.  gg  one  can  also  see  why  it  is 
necessary  to  focus  down,  i.  e.,  bring  the  object  and  objective  nearer  together  when 
the  tube  is  lengthened.  See  also  fig.  58. 

denomination  as  the  object.  In  9  mm.  there  are  90  tenths,  and  in  f 
there  are  4  tenths,  then  the  whole  length  of  the  image  is  90  +  4  =  94 
tenths  of  a  millimeter.  The  object  is  2  tenths  of  a  millimeter,  then 


CH.  I V  ]  MA  GN1FICA  TION  AND  MIC  ROME  TRY  109 

there  must  have  been  a  magnification  of  94  -r-  2  =  47  diameters  in 
order  to  produce  an  image  94  tenths  of  a  millimeter  long. 

Put  the  25  mm.  (i  in.,  C,  or  X  12)  ocular  in  place  of  one  of  37 
mm.  focus,  and  then  put  the  camera  lucida  in  position.  Measure  the 
size  of  the  image  with  dividers  and  a  rule  as  before.  The  power  will 
be  considerably  greater  than  when  the  low  ocular  was  used.  This  is 
because  the  virtual  image  (Fig.  21,  B3A3)  seen  with  the  high  ocular  is 
larger  than  the  one  seen  with  the  low  one.  The  real  image  (Fig.  21, 
A1!*1)  remains  nearly  the  same,  and  would  be  just  the  same  if  positive, 
par-focal  oculars  (§  37,  72,  note)  were  used. 

Lengthen  the  tube  of  the  microscope  50-60  mm.  by  pulling  out 
the  draw-tube.  Remove  the  camera  lucida,  and  focus,  then  replace 
the  camera  and  obtain  the  magnification.  It  will  be  greater  than  with 
the  shorter  tube.  This  is  because  the  real  image  (Fig.  99)  is  formed 
farther  from  the  objective  when  the  tube  is  lengthened,  and  the 
objective  must  be  brought  nearer  the  object.  The  law  is  :  The  size 
of  object  and  image  varies  directly  as  their  distance  from  the  center  of  the 
lens.  The  truth  of  this  statement  is  illustrated  by  Figs.  98  and  99. 

§  161.  Varying  the  Magnification  of  a  Compound  Micro- 
scope.— It  will  be  seen  from  the  above  experiments  (§  160)  that  in- 
dependently of  the  distance  at  which  the  microscopic  image  is 
measured  (§  162),  there  are  three  ways  of  varying  the  power  of  a 
compound  microscope.  These  are  named  below  in  the  order  of 
desirability. 

1 i )  By  using  a  higher  or  lower  objective. 

(2)  By  using  a  higher  or  lower  ocular. 

(3)  By  lengthening  or  shortening  the  tube  of  the  microscope  (Fig. 
99).* 

§  162.  Standard  Distance  of  250  Millimeters  at  which  the 
Virtual  Image  is  Measured. — For  obtaining  the  magnification  of 
both  the  simple  and  the  compound  microscope  the  directions  were  to 
measure  the  virtual  image  at  a  distance  of  250  millimeters.  This  is  not 


*  Amplifier. — In  addition  to  the  methods  of  varying  the  magnification  given 
in  \  161,  the  magnification  is  sometimes  increased  by  the  use  of  an  amplifier,  that 
is  a  diverging  lens  or  combination  placed  between  the  objective  and  ocular  and 
serving  to  give  the  image-forming  rays  from  the  objective  an  increased  divergence. 
An  effective  form  of  this  accessory  was  made  by  Tolles,  who  made  it  as  a  small 
achromatic  concavo-convex  lens  to  be  screwed  into  the  lower  end  of  the  draw-tube 
(frontispiece)  and  thus  but  a  short  distance  above  the  objective.  The  divergence 
given  to  the  rays  increases  the  size  of  the  real  image  about  two-fold. 


no 


MAGNIFICATION  AND  MICROMETRY 


[CH.  IV 


FIG.  100.  Figure  showing 
the  position  of  the  microscope, 
the  camera  lucida,  the  eye,  and 
the  difference  in  size  of  the  im- 
age depending  upon  the  dis- 
tance at  which  it  is  projected 
from  the  eye.  (a)  The  size  at 
25  cm.;  (b]  at 35  cm.,  (\  162). 


that  the  image  could  not  be  seen  and  measured  at  any  other  distance,  but 
because  some  standard  must  be  selected,  and  this  is  the  most  common 
one.  The  necessity  for  the  adoption  of  some  common  standard  will  be 
seen  at  a  glance  in  Fig.  100,  where  is  represented  graphically  the  fact 


FIG.  101.  Sectional  view 
of  the  Abbe  Camera  Lucida 
to  show  that  in  measuring 
the  standard  distance  of  250 
millimeters,  one  must  meas- 
ure along  the  axis  from  the 
point  P,  at  the  left  of  the 
prism,  to  the  mirror,  and 
from  the  mirror  to  the 
drawing  surface.  For  a 
full  explanation  of  this 
camera  lucida,  see  next 
chapter. 


FIG.  101. 

that  the  size  of  the  virtual  image  depends  directly  on  the  distance  at 
which  it  is  projected,  and  this  size  is  directly  proportional  to  the  ver- 
tical distance  from  the  apex  of  the  triangle,  of  which  it  forms  a  base. 
The  distance  of  250  millimeters  has  been  chosen  on  the  supposition 
that  it  is  the  distance  of  most  distinct  vision  for  the  normal  human  eye. 


CH,  IV\ 


MAGNIFICATION  AND  MICROMETRY 


III 


Demonstrate  the  difference  in  magnification  due  to  the  distance  at 
which  the  image  is  projected,  by  raising  the  microscope  so  that  the 
distance  will  be  350  millimeters,  then  lowering  to  150  millimeters. 

In  preparing  drawings  it  is  often  of  great  convenience  to  make 
them  at  a  distance  somewhat  less  or  somewhat  greater  than  the  stand- 
ard. In  such  a  case  the  magnification  must  be  determined  for  the 
special  distance.  (See  the  next  chapter,  §  181.) 

For  discussion  of  the  magnification  of  the  microscope,  see  :  Beale, 
pp.  41,  355  ;  Carpenter-Dallinger,  p.  288  ;  Nageli  and  Schwendener, 
p.  176  ;  Ranvier,  p.  29;  Robin,  p.  126  ;  Amer.  Soc.  Micrs. ,  1884,  p. 
183  ;  1889,  p.  22  ;  Amer.  Jour.  Arts  and  Sciences,  1890,  p.  50  ;  Jour. 
Roy.  Micr.  Soc.,  1888,  1889. 


OCULAR                                  OCULAR 
37  or  50  mm.                                   25  mm 

OBJECTIVE. 

TUBE 

IN 

TUBE 

OUT 
MM. 

TUBE 

IN 

TUBE 

OUT 
MM. 

OCULAR  MICROMETER 
VALUATION. 
TUBE  IN.    OUT  MM. 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

x    . 

X 

X 

X 

X 

X 

X 

X 

SIMPLE  MICROSCOPE.           X 

FIG.  102. 

§  163.  Table  of  Magnifications  and  of  the  Valuations  of  the 
Ocular  Micrometer.—  The  above  table  should  be  filled  out  by  each 
student.  In  using  it  for  Micrometry  and  Drawing  it  is  necessary  to  keep 
clearly  in  mind  the  exact  conditions  under  which  the  determinations  were 


112  MAGNIFICATION  AND  MICROMETRY  [CH.  IV 

made,  and  also  the  ways  in  which  variations  in  magnification  and  the  val- 
uation of  the  ocular  micrometer  may  be  produced  (§  161.  162,  172,  176). 

MICROMETRY 

§  164.  Micrometry  is  the  determination  of  the  size  of  objects  by 
the  aid  of  a  microscope. 

MICROMETRY   WITH    THE   SIMPLE    MICROSCOPE 

§  165.  With  a  simple  microscope  (A),  the  easiest  and  best  way 
is  to  use  dividers  and  then  with  the  simple  microscope  determine 
when  the  points  of  the  dividers  exactly  include  the  object.  The  spread 
of  the  dividers  is  then  obtained  as  above  (§  157).  This  amount  will 
be  the  actual  size  of  the  object,  as  the  microscope  was  only  used  in 
helping  to  see  when  the  divider  points  exactly  enclosed  the  object,  and 
then  for  reading  the  divisions  on  the  rule  in  getting  the  spread  of  the 
dividers. 

(B)  One  may  put  the  object  under  the  simple  microscope  and 
then,  as  in  determining  the  power  (§  156),  measure  the  image  at  the 
standard  distance.  If  the  size  of  the  image  so  measured  is  divided 
by  the  magnification  of  the  simple  microscope,  the  quotient  will  give 
the  actual  size  of  the  object. 

Use  a  fly's  wing  or  some  other  object  of  about  that  size,  and  try 
to  determine  the  width  in  the  two  ways  described  above.  If  all  the 
work  is  accurately  done  the  results  will  agree. 

MICROMETRY   WITH   THE   COMPOUND    MICROSCOPE 

There  are  several  ways  of  varying  excellence  for  obtaining  the  size 
of  objects  with  the  compound  microscope,  the  method  with  the  ocular 
micrometer  (§  175-176)  being  most  accurate. 

§  166.  Unit  of  Measure  in  Micrometry. — As  most  of  the  ob- 
jects measured  with  the  compound  microscope  are  smaller  than  any  of 
the  originally  named  divisions  of  the  meter,  and  the  common  or  decimal 
fractions  necessary  to  express  the  size  are  liable  to  be  unnecessarily 
cumbersome,  Harting,  in  his  work  on  the  microscope  (1859),  proposed 
the  one  thousandth  of  a  millimeter  (y^^  mm.  or  o.ooi  mm.)  or 
one  millionth  of  a  meter  (-nnriinnr  or  o.oooooi  meter)  as  the  unit.  He 
named  this  unit  micro-millimeter  and  designated  it  mmm.  In  1869, 
Listing  (Carl's  Repetorium  fur  Experimental-Physik,  Bd,  X,  p.  5) 


CH.  IV]  MAGN1FICA  TION  AND  MICROMETR  Y  113 

favored  the  thousandth  of  a  millimeter  as  unit  and  introduced  the  name 
Mikron  or  micrum.  In  English  it  is  most  often  written  Micron  (plural 
micra  or  microns,  pronunciation  Mik'r6n  or  Mik'r6n).  By  universal  con- 
sent the  sign  or  abbreviation  used  to  designate  it  is  the  Greek  //. 
Adopting  this  unit  and  sign,  one  would  express  five  thousandths  of  a 
millimeter  (T-<jVo  or  °-°°5  mm.)  thus,  5//.* 

\  167.  Micrometry  by  the  use  of  a  stage  micrometer  on  which  to  mount  the  ob- 
ject.— In  this  method  the  object  is  mounted  on  a  micrometer  and  then  put  under 
the  microscope,  and  the  number  of  spaces  covered  by  the  object  is  read  off  directly. 
It  is  exactly  like  putting  any  large  object  on  a  rule  and  seeing  how  many  spaces  of 
the  rule  it  covers.  The  defect  in  the  method  is  that  it  is  impossible  to  properly 
arrange  objects  on  the  micrometer.  Unless  the  objects  are  circular  in  outline  they 
are  liable  to  be  oblique  in  position,  and  in  every  case  the  end  or  edges  of  the  object 
may  be  in  the  middle  of  a  space  instead  of  against  one  of  the  lines,  consequently 
the  size  must  be  estimated  or  guessed  at  rather  than  really  measured. 

§  1 68 .  Micrometry  by  dividing  the  size  of  the  image  by  the  magnifica- 
tion of  the  microscope. — For  example,  employ  the  3  mm.  (^6  in.)  objective, 
25  mm.  (i  in.)  ocular,  and  a  Necturus'  red  blood-corpuscle  preparation 
as  object.  Obtain  the  size  of  the  image  of  the  long  and  short  axes 
of  three  corpuscles  with  the  camera  lucida  and  dividers,  exactly  as  in 
obtaining  the  magnification  of  the  microscope  (§  160).  Divide  the  size 
of  the  image  in  each  case  by  the  magnification,  and  the  result  will  be 
the  actual  size  of  the  blood-corpuscles.  Thus,  suppose  the  image  of  the 
long  axis  of  the  corpuscle  is  18  mm.  and  the  magnification  of  the  micro- 
scope 400  diameters  (§  154),  then  the  actual  length  of  this  long  axis  of 
the  corpuscle  is  18  mm.-r-  400=0.045  mm.  or  45^  (§  166). 


FIG.  103.     Preparation  of  blood  with  a 
ring  around  a  group  of  blood  corpuscles. 


As  the  same  three  blood-corpuscles  are  to  be  measured  in  three 
ways,  it  is  an  advantage  to  put  a  delicate  ring  around  a  group  of  three 
or  more  corpuscles,  and  make  a  sketch  of  the  whole  enclosed  group, 

*The  term  micromillimeter,  abbreviation  mmm.,  is  very  cumbersome,  and  be- 
sides is  entirely  inappropriate  since  the  adoption  of  the  definite  meanings  for  the 
prefixes  micro  and  mega,  meaning  respectively  one-millionth  and  one  million 
times  the  unit  before  which  it  is  placed.  A  micromillimeter  would  then  mean 
one- millionth  of  a  millimeter,  not  one-thousandth.  The  term  micron  has  been 
adopted  by  the  great  microscopical  societies,  the  international  commission  on 
weights  and  measures,  and  by  original  investigators,  and  is,  in  the  opinion  of  the 
writer,  the  best  term  to  employ.  Jour.  Roy.  Micr.  Soc.,  1888,  p.  502  ;  Nature, 
Vol.  XXXVII  (1888),  p.  388. 


1 14  MAGNIFICA TION  AND  M1CROMETRY  [£7/.  IV 

marking  on  the  sketch  the  corpuscles  measured  (Figs.  61-66).  The 
different  corpuscles  vary  considerably  in  size,  so  that  accurate  com- 
parison of  different  methods  of  measurement  can  only  be  made  when 
the  same  corpuscles  are  measured  in  each  of  the  ways. 

§  169.  Micrometry  by  the  use  of  a  Stage  Micrometer  and  a 
Camera  Lucida. — Employ  the  same  object,  objective  and  ocular  as 
before.  Put  the  camera  lucida  in  position,  and  with  a.  lead  pencil 
make  dots  on  the  paper  at  the  limits  of  the  image  of  the  blood- 
corpuscle.  Measure  the  same  three  that  were  measured  in  §  168. 

Remove  the  object,  place  the  stage  micrometer  under  the  micro- 
scope, focus  well,  and  draw  the  lines  of  the  stage  micrometer  so  as  to 
include  the  dots  representing  the  limits  of  the  part  of  the  image  to  be 
measured.  As  the  value  of  the  spaces  on  the  stage  micrometer  is 
known,  the  size  of  the  object  is  determined  by  the  number  of  spaces 
of  the  micrometer  required  to  include  it. 

This  simply  enables  one  to  put  the  image  of  a  fine  rule  on  the 
image  of  a  microscopic  object.  It  is  theoretically  an  excellent  method, 
and  nearly  the  same  as  measuring  the  spread  of  the  dividers  with  a 
simple  microscope  (§  157,  176). 

OCULAR    MICROMETER 

§  170.  Ocular  Micrometer,  Eye-Piece  Micrometer. — This, 
as  the  name  implies,  is  a  micrometer  to  be  used  with  the  ocular.  It  is 
a  micrometer  on  glass,  and  the  lines  are  sufficiently  coarse  to  be  clearly 
seen  by  the  ocular.  The  lines  should  be  equidistant  and  about  y^th  or 
2^th  mm.  apart,  and  every  fifth  line  should  be  longer  and  heavier  to 
facilitate  counting.  If  the  micrometer  is  ruled  in  squares  (net  micro- 
meter} it  will  be  very  convenient  for  many  purposes. 

The  ocular  micrometer  is  placed  in  the  ocular,  no  matter  what  the 
form  of  the  ocular  (i.  e.,  whether  positive  or  negative)  at  the  level  at 
which  the  real  image  is  formed  by  the  objective,  and  the  image  appears 
to  be  immediately  upon  or  under  the  ocular  micrometer,  and  hence 
the  number  of  spaces  on  the  ocular  micrometer  required  to  measure 
the  real  image  may  be  read  off  directly.  This,  however,  is  measuring 
the  size  of  the  real  image,  and  the  actual  size  of  the  object  can  only  be 
determined  by  determining  the  ratio  between  the  size  of  the  real  image 
and  the  object.  In  other  words,  it  is  necessary  to  get  the  valuation  of 
the  ocular  micrometer  in  terms  of  a  stage  micrometer. 

§  171.  Valuation  of  the  Ocular  Micrometer. — This  is  the 
value  of  the  divisions  of. the  ocular  micrometer  for  the  purposes  of 


CH. 


MAGNIFICATION  AND  MICROMETRY 


micrometry,  and  is  entirely  relative,  depending  upon  the  magnification 
of  the  real  image  formed  by  the  objective,  consequently  it  changes 
with  every  change  in  the  magnification  of  the  real  image,  and  must  be 
especially  determined  for  every  optical  combination  (i.  e.,  objective  and 
ocular),  and  for  every  change  in  the  length  of  the  tube  of  the  micro- 
scope. That  is,  it  is  necessary  to  determine  the  ocular  micrometer  val- 
uation for  every  condition  modifying  the  real  image  of  the  microscope 

(§  161). 

Any  Huygenian  ocular  (Fig.  30)  may,  however,  be  used  as  a  micrometer  ocu- 
lar by  placing  the  ocular  micrometer  at  the  level  of  the  ocular  diaphragm,  where 
the  real  image  is  formed.  If  there  is  a  slit  in  the  side  of  the  ocular  ,  and  the 
ocular  micrometer  is  mounted  in  some  way  it  may  be  introduced  through  the 
opening  in  the  side.  When  no  side  opening  exists  the  mounting  of  the  eye-lens 
may  be  unscrewed  and  the  ocular  micrometer,  if  on  a  cover-glass  can  be  laid  on 
the  upper  side  of  the  ocular  diaphragm. 


FIG.  104.  FIG.  105. 

FIGS.  104-105.  Ocular  Micrometer  with  movable  scale.  Fig.  104  is  a  side  view 
of  the  octilar  while  Fig.  105  gives  a  sectional  end  view,  and  shows  the  ocular  mi- 
crometer in  position .  In  both  the  screw  which  moves  the  micrometer  is  shown  at 
the  left.  (Bausch  &  Lomb  Opt.  Co.) 

§  172.  Obtaining  the  Ocular  Micrometer  Valuation  for  an 
Ocular  Micrometer  with  Fixed  Lines  (Figs.  104-105). — Use  the 
stage  micrometer  as  object.  Light  the  field  well  and  look  into  the 
microscope.  The  lines  of  the  ocular  micrometer  should  be  very  sharply 
defined.  If  they  are  not,  raise  or  lower  the  eye-lens  to  make  them  so  ; 
that  is,  focus  as  with  the  simple  magnifier. 

When  the  lines  of  the  ocular  micrometer  are  distinct,  focus  the 
microscope  (§72,  74,  75)  for  the  stage  micrometer.  The  image  of  the 
stage  micrometer  will  appear  to  be  directly  under  or  upon  the  ocular 
micrometer. 

Make  the  lines  of  the  two  micrometers  parallel  by  rotating  the  ocular 
or  changing  the  position  of  the  stage  micrometer  or  both  if  necessary, 


Ii6  MAGNIFICATION  AND  MICROMETRY  \_CH.  IV 

and  then  make  any  two  lines  of  the  stage  micrometer  coincide  with 
any  two  on  the  ocular  micrometer.  To  do  this  it  may  be  necessary  to 
pull  out  the  draw- tube  a  greater  or  less  distance.  See  how  many 
spaces  are  included  in  each  of  the  micrometers. 

Divide  the  value  of  the  included  space  or  spaces  on  the  stage  mi- 
crometer by  the  number  of  divisions  on  the  ocular  micrometer  required 
to  include  them,  and  the  quotient  so  obtained  will  give  the  valuation  of 
the  ocular  micrometer  in  fractions  of  the  unit  of  measure  of  the  stage 
micrometer.  For  example,  suppose  the  millimeter  is  taken  as  the  unit 
for  the  stage  micrometer  and  this  unit  is  divided  into  spaces  of  y^th  and 
y-Q-g-th  millimeter.  If  now,  with  a  given  optical  combination  and  tube- 
length,  it  requires  10  spaces  on  the  ocular  micrometer  to  include  the 
real  image  of  yVth  millimeter  on  the  stage  micrometer,  obviously  one 
space  on  the  ocular  micrometer  would  include  only  one-tenth  as  much, 
or  yVth  mm.  -=-10  =  y-J^th  mm.  That  is,  each  space  on  the  ocular  mi- 
crometer would  include  y-^oth  of  a  millimeter  on  the  stage  micrometer, 
or  y-Jijth  millimeter  of  the  length  of  any  object  under  the  microscope, 
the  conditions  remaining  the  same.  Or,  in  other  words,  it  would  re- 
quire 100  spaces  on  the  ocular  micrometer  to  include  i  millimeter  on  the 
stage  micrometer,  then  as  before,  i  space  of  the  ocular  micrometer  would 
have  a  valuation  of  y^-Q-th  millimeter  for  the  purposes  of  micrometry. 
The  size  of  any  minute  object  may  be  determined  by  multiplying  this 
valuation  of  one  space  by  the  number  of  spaces  required  to  include  it. 
For  example,  suppose  the  fly's  wing  or  some  part  of  it  covered  8  spaces 
on  the  ocular  micrometer,  it  would  be  known  that  the  real  size  of  the 
part  measured  is  y^th  mm.  x  8  =  y-fo-  mm.  or  80  ju.  (§  166).  See 
Mark,  Jour.  Applied  Microscopy,  Vol.  I,  p.  4. 

§  173.  Micrometry  with  the  Ocular  Micrometer. — Use  the  3 
mm.  (-|-  in. )  objective  with  the  preparation  of  Necturus  blood-corpuscles 
as  object.  Make  certain  that  the  tube  of  the  microscope  is  of  the  same 
length  as  when  determining  the  ocular  micrometer  valuation.  In  a 
word,  be  sure  that  all  the  conditions  are  exactly  as  when  the  valuation 
was  determined,  then  put  the  preparation  under  the  microscope  and 
find  the  same  three  red  corpuscles  that  were  measured  in  the  other 
ways  (§168-169). 

Count  the  divisions  on  the  ocular  micrometer  required  to  enclose 
or  measure  the  long  and  the  short  axis  of  each  of  the  three  corpuscles, 
multiply  the  number  of  spaces  in  both  cases  by  the  valuation  of  the 
ocular  micrometer  for  this  objective,  tube-length  and  ocular,  and  the 
results  will  represent  the  actual  length  of  the  axes  of  the  corpuscles 
in  each  case. 


CH. 


MAGNIFICATION  AND  MICROMETRY 


117 


The  same  corpuscle  is,  of  course,  of  the  same  actual  size,  when 
measured  in  each  of  the  three  ways,  so  that  if  the  methods  are  correct 
and  the  work  carefully  enough  done,  the  same  results  should  be  ob- 
tained by  each  method.  (§  176).* 

FIG.  1 06.  Ocular  Screw- Micrometer  with 
compensation  ocular  X  6.  The  upper  figure 
shows  a  sectional  view  of  the  ocular  and  the 
screw  for  moving  the  micrometer  at  the  right. 
At  the  left  is  shown  a  clamping  screw  to 
fasten  the  ocular  to  the  upper  part  of  the  mi- 
croscope tube.  Below  is  a  face  view,  showing 
the  graduation  on  the  wheel.  An  ocular 
micrometer  like  this  is  in  general  like  the 
cob-web  micrometer  and  may  be  used  for 
measuring  objects  of  varying  sizes  very  accu- 
rately. With  the  ordinary  ocular  micrometer 
very  small  objects  frequently  fill  but  a  part  of 
an  interval  of  the  micrometer,  but  with  this 
the  movable  cross  lines  traverse  the  object  (or 
rather  its  real  image}  regardless  of  the  minute- 
ness of  the  object.  (Zeiss*  Catalog}. 

\  174.  Obtaining  the  Valuation  of  the  Screw  or  Filar  Micrometer. — This 
micrometer  (Fig.  106-107)  usually  consists  of  a  Ramsden's  ocular  and  cross  lines. 
As  seen  in  Fig.  107^  there  are  three  lines.  The  horizontal  and  one  vertical  line 
are  fixed.  One  vertical  line  may  be  moved  by  the  screw  back  and  forth  across  the 
field. 

For  obtaining  the  valuation  of  this  ocular  micrometer  an  accurate  stage  mi- 
crometer must  be  used.  Carefully  focus  the  Tiy^th  mm.  spaces.  The  lines  of  the 
ocular  micrometer  should  also  be  sharp.  If  they  are  not  focus  them  by  moving 
the  top  of  the  ocular  up  or  down  ($  172).  Make  the  vertical  lines  of  the  filar  mi- 
crometer parallel  with  the  lines  of  the  stage  micrometer.  Take  the  precautions 
regarding  the  width  of  the  stage  micrometer  lines  given  in  $  176  (see  also  Fig. 
108).  Note  the  position  of  the  graduated  wheel  and  of  the  teeth  of  the  recording 
comb,  and  then  rotate  the  wheel  until  the  movable  line  traverses  one  space  on  the 
stage  micrometer.  Each  tooth  of  the  recording  comb  indicates  a  total  revolution 
of  the  wheel,  and  by  noting  the  number  of  teeth  required  and  the  graduations  on 
the  wheel,  the  revolutions  and  parts  of  revolution  required  to  measure  the  j^ih 
mm,  of  the  stage  micrometer  can  be  easily  noted.  Measure  in  like  manner  4  or  5 
spaces  and  get  the  average.  Suppose  this  average  is  i#th  revolutions  or  125 
graduations  on  the  wheel,  to  measure  the  Tfoth  mm.  or  IQJU  (see  %  166),  then  one 
of  the  graduations  on  the  wheel  would  measure  IQJU  divided  by  I25=.o8//.  In 
using  this  valuation  for  actual  measurement,  the  tube  of  the  microscope  and  the 
objective  must  be  exactly  as  when  obtaining  the  valuation  (see  \  175). 

Example  of  Measurement. — Suppose  one  uses  the  red  blood  corpuscles  of  a  dog 
or  monkey,  etc.,  every  condition  being  as  when  the  valuation  was  determined,  one 
notes  very  accurately  how  many  of  the  graduations  on  the  wheel  are  required  to 
make  the  movable  line  traverse  the  object  from  edge  to  edge.  Suppose  it  requires 


u8 


MAGNIFICATION  AND  MICROMETRY 


[CH.IV 


94  of  the  graduations  to  measure  the  diameter,  the  actual  size  of  the  corpuscle 
would  be  94X.o8  ju  =  7.52^. 

The  advantage  of  the  filar  micrometer  is  that  the  valuation  of  one  graduation 
being  so  small,  even  the  smallest  object  to  be  measured  would  require  several 
graduations  to  measure  it.  In  ocular  micrometers  with  fixed  lines,  small  objects 
like  bacteria  might  not  fill  even  one  space,  therefore  estimations,  not  measure- 
ments, must  be  made.  For  large  objects,  like  most  of  the  tissue  elements,  the 
ocular  micrometers  with  fixed  lines  answer  very  well,  for  the  part  which  must  be 
estimated  is  relatively  small  and  the  chance  of  error  is  correspondingly  small. 


FIG.  107.  Filar  Ocular  Micrometer  with  Field  A  (Bausch  &  Lomb,  Optical  Co.}. 
§  175.  Varying  the  Ocular  Micrometer  Valuation. — Any 
change  in  the  objective,  the  ocular  or  the  tube-length  of  the  microscope, 
that  is  to  say,  any  change  in  the  size  of  the  real  image,  produces  a  cor- 
responding change  in  the  ocular  micrometer  valuation  (§  161,  171, 
176). 

\  176.  Remarks  on  Micrometry. — In  using  adjustable  objectives  (§24,  103), 
the  magnification  of  the  objective  varies  with  the  position  of  the  adjusting  collar, 
being  greater  when  the  adjustment  is  closed  as  for  thick  cover-glasses  than  when 
open,  as  for  thin  ones.  This  variation  in  the  magnification  of  the  objective  pro- 
duces a  corresponding  change  in  the  magnification  of  the  entire  microscope,  and 
the  ocular  micrometer  valuation — therefore  it  is  necessary  to  determine  the  mag- 
nification and  ocular  micrometer  valuation  for  each  position  of  the  adjusting 
collar. 

While  the  principles  of  micrometry  are  simple,  it  is  very  difficult  to  get  the 
exact  size  of  microscopic  objects.  This  is  due  to  the  lack  of  perfection  and  uni- 

*There  are  three  ways  of  using  the  ocular  micrometer,  or  of  arriving  at  the  size 
of  the  objects  measured  with  it : 

(A)  By  finding  the  value  of  a  division  of  the  ocular  micrometer  for  each  optical 
combination  and  tube-length  used,  and  employing  this  valuation  as  a  multiplier. 
This  is  the  method  given  in  the  text,  and  ;the  one  most  frequently  employed. 
Thus,  suppose  with  a  given  optical  combination  and  tube-length  it  required  five 
divisions  on  the  ocular  micrometer  to  include  the  image  of  r20ths  millimeter  of  the 
stage  micrometer,  then  obviously  one  space  on  the  ocular  micrometer  would  in- 
clude ith  of  T2(jths  nvm.  or  ^th  mm.;  the  size  of  any  unknown  object  under  the 


CH.  IV\  MA  GNIFICA  TION  AND  MICRO  ME  TRY  119 

fortuity  of  micrometers,  and  the  difficulty  of  determining  the  exact  limits  of  the 
object  to  be  measured.  Hence,  all  microscopic  measurements  are  only  approxi- 
mately correct,  the  error  lessening  with  the  increasing  perfection  of  the  apparatus 
and  the  skill  of  the  observer. 


microscope  would  be  obtained  by  multiplying  the  number  of  divisions  on  the 
ocular  micrometer  required  to  include  its  image  by  the  value  of  one  space,  or  in 
this  case,  ^th  mm.  Suppose  some  object,  as  the  fly's  wing,  required  15  spaces  of 
the  ocular  micrometer  to  include  some  part  of  it,  then  the  actual  size  of  this  part 
of  the  wing  would  be  15  X  -h  —  fths,  or  0.6  mm. 

(B)  By  finding  the  number  of  divisions  on  the  ocular  micrometer  required  to 
include  the  image  of  an  entire  millimeter  of  the  stage  micrometer,  and  using  this 
number  as  a  divisor.     This  number  is  also  sometimes  called  the  ocular  micrometer 
ratio.     Taking  the  same  case  as  in  (A),  suppose  five  divisions  of  the  ocular  mi- 
crometer are  required  to  include  the  image  of  T25ths  mm.,  on  the  stage  micrometer, 
then  evidently  it  would  require  5  -j-  T%  —  25  divisions  on  the  ocular  micrometer  to 
include  a  whole  millimeter  on  the  stage  micrometer,  and  the  number  of  divisions 
of  the  ocular  micrometer  required  to  measure  an  object  divided  by  25  would  give 
the  actual  size  of  the  object  in  millimeters  or  in  a  fraction  of  a  millimeter.     Thus, 
suppose  it  required  15  divisions  of  the  ocular  micrometer  to  include  the  image  of 
some  part  of  the  fly's  wing,  the  actual  size  of  the  part  included  would  be  15  H-  25 
—  |  or  0.6  mm.     This  method  is  really  exactly  like  the  one  in  (A),  for  dividing  by 
25  is  the  same  as  multiplying  by  ^th. 

(C)  By  having  the  ocular  micrometer  ruled  in  millimeters  and  divisions  of  a 
millimeter,  and  then  getting  the  size  of  the  real  image  in  millimeters.     In  employ- 
ing this  method  a  stage  micrometer  is  used  as  object  and  the  size  of  the  image  of 
one  or  more  divisions  is  measured  by  the  ocular  micrometer,   thus  :     Suppose  the 
stage  micrometer  is  ruled  in  T^th  andy^th  mm.  and  the  ocular  micrometer  is  ruled 
in  millimeters  and  TVth  mm.     Taking  y2ffth  mm.  on  the  stage  micrometer  as  object, 
as  in  the  other  cases,  suppose  it  requires  10  of  the  y^th  mm.   spaces  or   I   mm.  to 
measure  the  real  image,  then  the  real  image  must  be  magnified  y$-=-y2^  =  5  diame- 
ters, that  is,  the  real  image  is  five  times  as  great  in  length  as  the  object,  and  the 
size  of  an  object  may  be  determined  by  putting  it  under  the  microscope  and  getting 
the  size  of  the  real  image  in  millimeters  with  the  ocular  micrometer  and  dividing 
it  by  the  magnification  of  the  real  image,  which  in  this  case  is  5  diameters. 

Use  the  fly's  wing  as  object,  as  in  the  other  cases,  and  measure  the  image  of 
the  same  part.  Suppose  that  it  required  30  of  the  y1^  mm.  divisions  =  f§  mm.  or  3 
mm.  to  include  the  image  of  the  part  measured,  then  evidently  the  actual  size  of 
the  part  measured  is  3  mm,  -H  5  =  f  mm.,  the  same  result  as  in  the  other  cases. 

In  comparing  these  methods  it  will  be  seen  that  in  the  first  two  (A  and  B )  the 
ocular  micrometer  may  be  simply  ruled  with  equidistant  lines  without  regard  to 
the  absolute  size  in  millimeters  or  inches  of  the  spaces.  In  the  last  method  the 
ocular  micrometer  must  have  its  spaces  some  known  division  of  a  millimeter  or 
inch.  In  the  first  two  methods  only  one  standard  of  measure  is  required,  viz,  the 
stage  micrometer  ;  in  the  last  method  two  standards  must  be  used, — a  stage  mi- 
crometer and  an  ocular  micrometer.  Of  course,  the  ocular  micrometer  in  the  first 
two  cases  must  have  the  lines  equidistant  as  well  as  in  the  last  case,  but  ruling 
lines  equidistant  is  quite  a  different  matter  from  getting  them  an  exact  division  of 
a  millimeter  or  of  an  inch  apart. 


120 


MAGNIFICATION  AND  MICROMETRY 


[CH.  IV 


A  difficulty  when  one  is  using  high  powers  is  the  width  of  the  lines  of  the 
micrometer.  If  the  micrometer  is  perfectly  accurate  half  the  width  of  each  line 
belongs  to  the  contiguous  spaces,  hence  one  should  measure  the  image  of  the  space 
from  the  centers  of  the  lines  bordering  the  space,  or  as  this  is  somewhat  difficult 
in  using  the  ocular  micrometer,  one  may  measure  from  the  inside  of  one  border- 
ing line  and  from  the  outside  of  the  other.  If  the  lines  are  of  equal  width  this  is 
as  accurate  as  measuring  from  the  center  of  the  lines.  Evidently  it  would  not  be 
right  to  measure  from  either  the  inside  or  the  outside  of  both  lines  (Fig.  108). 

It  is  also  necessary  in  micrometry  to  use  an  objective  of  sufficient  power  to 
enable  one  to  see  all  the  details  of  an  object  with  great  distinctness.  The  necessity 
of  using  sufficient  amplification  in  micrometry  has  been  especially  remarked  upon 
by  Richardson,  Monthly  Micr.  Jour.,  1874,  1875,  ;  Rogers,  Proc.  Amer.  Soc.  Micro- 
scopists,  1882,  p.  239;  Ewell,  North  American  Pract.,  1890,  pp.  97,  173. 

FIG.  108.   The  appearance  of  the  coarse 

A  B  stage  micrometer  and  of  the  fine  ocular  mi- 

crometer lines  when  using  a  high  objective. 

(A}.  The  method  of  measuring  the 
spaces  by  ptdting  the  fine  ocular  mi- 
crometer lines  opposite  the  center  of  the 
coarse  stage  micrometer  lines. 

( B) .  Method  of  measuring  the  spaces 
of  the  stage  micrometer  by  putting  one 
line  of  the  ocular  micrometer  (o.  m.}  at  the 
inside  and  one  at  the  outside  of  the  coarse 
stage  micrometer  lines  (s.  m.). 

FIG.  108. 

As  to  the  limit  of  accuracy  in  micrometry,  one  who  has  justly  earned  the  right 
to  speak  with  authority  expresses  himself  as  follows:  ^  I  assume  that  0.2  n  is  the 
limit  of  precision  in  microscopic  measures  beyond  which  it  is  impossible  to  go  with 
certainty."  W.  A.  Rogers  Proc.  Amer.  Soc.  Micrs.,  1883,  p.  198. 

In  comparing  the  methods  of  micrometry  with  the  compound  microscope  given 
above  ($  167,  168,  169,  175),  the  one  given  in  \  167  is  impracticable,  that  given  in 
\  168  is  open  to  the  objection  that  two  standards  are  required, — the  stage  microme- 
ter, and  the  steel  rule ;  it  is  open  to  the  further  objection  that  several  different 
operations  are  necessary,  each  operation  adding  to  the  probability  of  error.  Theoret- 
ically the  method  given  in  \  169  is  good,  but  it  is  open  to  the  very  serious  objection 
in  practice  that  it  requires  so  many  operations  which  are  especially  liable  to  intro- 
duce errors.  The  method  that  experience  has  found  most  safe  and  expeditious, 
and  applicable  to  all  objects,  is  the  method  with  the  ocular  micrometer.  If  the 
valuation  of  the  ocular  micrometer  has  been  accurately  determined,  then  the  only 
difficulty  is  in  deciding  on  the  exact  limits  of  the  objects  to  be  measured  and  so 
arranging  the  ocular  micrometer  that  these  limits  are  inclosed  by  some  divisions  of 
the  micrometer.  Where  the  object  is  not  exactly  included  by  whole  spaces  on  the 
ocular  micrometer,  the  chance  of  error  comes  in,  in  estimating  just  how  far  into  a 
space  the  object  reaches  on  the  side  not  in  contact  with  one  of  the  micrometer 


[_CH. 


MA  GNIFICA  TION  AND  MICRO  ME  TR  Y 


121 


lines.  If  the  ocular  micrometer  has  some  quite  narrow  spaces,  and  others  consid- 
erably larger,  one  can  nearly  always  manage  to  exactly  include  the  object  by  some 
two  lines.  The  ocular  screw  micrometers  (Fig.  106-107)  obviates  this  entirely  as 
the  cross  hair  or  lines  traverse  the  object  or  its  real  image,  and  whether  this 
distance  be  great  or  small  it  can  be  read  off  on  the  graduated  wheel,  and  no 
estimation  or  guess  work  is  necessary. 

For  those  especially  interested  in  micrometry,  as  in  its  relation  to  medical 
jurisprudence,  the  following  references  are  recommended.  These  articles  consider 
the  problem  in  a  scientific  as  well  as  a  practical  spirit  :  The  papers  of  Prof.  Wm. 
A.  Rogers  on  micrometers  and  micrometry,  in  the  Amer.  Quar.  Micr.  Jour.,  Vol. 
!>  PP-  97  >  2°8  ;  Proceedings  Amer.  Soc.  Microscopists,  1882,  1883,  1887.  Dr.  M. 
D.  Ewell,  Proc.  Amer.  Soc.  Micrs.,  1890  ;  The  Microscope,  1889,  pp.  43-45  ;  North 
Amer.  Pract.,  1890,  pp.  97,  173.  Dr.  J.  J.  Woodward,  Amer.  Jour,  of  the  Med. 
Sci.,  1875.  M.  C.  White,  Article  "Blood-stains,"  Ref.  Hand-Book  Med.  Sciences, 
1885.  Medico-Legal  Journal,  Vol.  XII.  For  the  change  in  magnification  due  to 
a  change  in  the  adjustment  of  adjustable  objectives,  see  Jour.  Roy.  Micr.  Soc., 
iSSo,  p.  702  ;  Amer.  Monthly  Micr.  Jour.,  1880,  p.  67.  Carpenter-Dallinger,  p.  270. 

If  one  consults  the  medico-legal  journals  ;  the  microscopical  journals,  the 
Index  Medicus,  and  the  Index  Catalog  of  the  Library  of  the  Surgeon  General's 
Office,  under  Micrometry,  Blood,  and  Jurisprudence,  he  can  get  on  track  of  the 
main  work  which  has  been  and  is  being  done. 


f  in. 


Dry  objectives  of  16  mm.  (f  z«. ),  4.  mm.  (i  in.)  and  homogeneous  immersion 
objective  0/2  mm.  (TV  in. )  in  their  mountings.     (Bausch  &  Lomb  Opt.  Co.). 


CHAPTER  V 


DRAWING   WITH   THE   MICROSCOPE 


APPARATUS   AND    MATERIAL   FOR    THIS   CHAPTER 

Microscope,  Abbe   and   Wollaston's   camera  lucidas,  drawing  board,  thumb 
tacks,  pencils,  paper,  and  microscope  screen,  (Fig.  59),  microscopic  preparations. 

DRAWING   MICROSCOPIC   OBJECTS 

§  177.  Microscopic  objects  may  be  drawn  free-hand  directly  from 
the  microscope,  but  in  this  way  a  picture  giving  only  the  general  ap- 
pearance and  relations  of  parts  is  obtained.  For  pictures  which  shall 
have  all  the  parts  of  the  object  in  true  proportions  and  relations,  it  is 
necessary  to  obtain  an  exact  outline  of  the  image  of  the  object,  and  to 
locate  in  this  outline  all  the  principal  details  of  structure.  It  is  then 
possible  to  complete  the  picture  free-hand  from  the  appearance  of  the 
object  under  the  microscope.  The  appliance  used  in  obtaining  out- 
lines, etc.,  of  the  microscope  image  is  known  as  a  camera  lucida. 

§  178.  Camera  Lucida. — This  "is  an  optical  apparatus  for  en- 
abling one  to  see  objects  in  greatly  different  situations,  as  if  in  one 
field  of  vision,  and  with  the  same  eye.  In  other  words  it  is  an  optical 
device  for  superimposing  or  combining  two  fields  of  view  in  one  eye. 

As  applied  to  the  microscope,  it  causes  the  magnified  virtual  im- 
age of  the  object  under  the  microscope  to  appear  as  if  projected  upon 
the  table  or  drawing  board,  where  it  is  visible  with  the  drawing  paper, 
pencil,  dividers,  etc.,  by  the  same  eye,  and  in  the  same  field  of  vision. 
The  microscopic  image  appears  like  a  picture  on  the  drawing  paper 
(see  note  to  §  181).  This  is  accomplished  in  two  distinct  ways  : 

(A)  By  a  camera  lucida  reflecting  the  rays  from  the  microscope  so 
that  their  direction  when  they  reach  the  eye  coincides  with  that  of  the 
rays  from  the  drawing  paper,  pencil,  etc.  In  some  of  the  camera 
lucidas  from  this  group  (Wollaston's,  Fig.  112),  the  rays  are  reflected 
twice,  and  the  image  appears  as  when  looking  directly  into  the  micro- 
scope. In  others  the  rays  are  reflected  but  once,  and  the  image  has 
the  inversion  produced  by  a  plane  mirror.  For  drawing  purposes  this 


CH.  V] 


DRA  WING  WITH  THE  MICROSCOPE 


123 


inversion  is  a  great  objection,  as  it  is  necessary  to  similarly  invert   all 
the  details  added  free-hand. 

(B)  By  a  camera  lucida  reflecting  the  rays  of  light  from  the  draw- 
ing paper,  etc. ,  so  that  their  direction  when  they  reach  the  eye  coin- 
cides with  the  direction  of  the  rays  from  the  microscope  (Fig.  58,  109). 
In  all  of  the  camera  lucidas  of  this  group,  the  rays  from  the  paper  are 
twice  reflected  and  no  inversion  appears. 


FIG.  no. 


FIG.  in. 


FIG.  109. 


FIG.  109.  Abbe  Camera  Lucida  with 
the  mirror  at  45°,  the  drawing  surface 
horizontal,  and  the  microscope  vertical. 

Axis,  Axis.  Axial  ray  from  the  mi- 
croscope and  from  the  drawing  surface. 
A,  B.  Marginal  rays  of  the  field  on  the 
drawing  surface,  a  b.  Sectional  view  of 
the  silvered  surf  ace  on  the  upper  of  the  tri- 
angular prisms  composing  the  cubical 
prism  ( P) .  The  silvered  surface  is  shown 


as  incomplete  in  the  center,  thus  giving  passage  to  the  rays  from  the  microscope 

Foot.     Foot  or  base  of  the  microscope. 

G.  Smoked  glass  seen  in  section.  It  is  placed  between  the  mirror  and  the 
prism  to  reduce  the  light  from  the  drawing  surface. 

Mirror.  The  mirror  of  the  camera  lucida.  A  quadrant  (Q)  has  been  added 
to  indicate  the  angle  of  inclination  of  the  mirror,  which  in  this  case  is  45°. 

Ocular.  Ocular  of  the  microscope  over  which  the  prism  of  the  camera  lucida 
is  placed. 

P,  P.     Drawing  pencil  and  the  cubical  prism  over  the  ocular. 

FIG.  no.  Geometrical  figure  showing  the  angles  made  by  the  axial  ray  with 
the  drawing  surface  and  the  mirror. 

A,  B.     The  drawing  surface. 

FIG.  in.  Ocular  showing  eye-point,  E.  P.  It  is  at  this  point  both  horizontally 
and  vertically  that  the  hole  in  the  silvered  surface  should  be  placed  (|  182). 


124  DRAWING   WITH  THE  MICROSCOPE  [CH.  V 

The  better  forms  of  camera  lucidas  (Wollaston's,  Grunow's,  Abbe's, 
etc.),  may  be  used  for  drawing  both  with  low  and  with  high  powers. 
Some  require  the  microscope  to  be  inclined  (Fig.  TOO)  while  others  are 
designed  to  be  used  on  the  microscope  in  a  vertical  position.  As  in 
biological  work,  it  is  often  necessary  to  have  the  microscope  vertical, 
the  form  for  a  vertical  microscope  is  to  be  preferred  ;  but  see  Figs.  115- 

116. 

§   179.      Avoidance    of   Distortion. — In   order  that  the  pictiire 

drawn  by  the  aid  of  a  camera  lucida  may  not  be  distorted,  it  is  necessary 
that  the  axial  ray  from  the  image  on  the  drawing  surface  shall  be  at  right 
angles  to  the  drawing  surface  (Figs.  112,  114.) 

$  180.  Wollaston's  Camera  Lucida. — This  is  a  quadrangular  prism  of  glass 
put  in  the  path  of  the  rays  from  the  microscope,  and  it  serves  to  change  the 
direction  of  the  axial  ray  90  degrees.  In  using  it  the  microscope  is  made  horizon- 
tal, and  the  rays  from  the  microscope  enter  one-half  of  the  pupil  while  rays  from 
the  drawing  surface  enter  the  other  half  of  the  pupil.  As  seen  in  the  figure  (Fig. 
112),  the  fields  partly  overlap,  and  where  they  do  so  overlap,  pencil  or  dividers 
and  microscopic  image  can  be  seen  together. 

In  drawing  or  using  the  dividers  with  the  Wollaston  camera  lucida  it  is  neces- 
sary to  have  the  field  of  the  microscope  and  the  drawing  surface  about  equally 
lighted.  If  the  drawing  surface  is  too  brilliantly  lighted  the  pencil  or  dividers 
may  be  seen  very  clearly,  but  the  microscopic  image  will  be  obscure.  On  the 
other  hand,  if  the  field  of  the  microscope  has  too  much  light  the  microscopic 
image  will  be  very  definite,  but  the  pencil  or  dividers  will  not  be  visible.  It  is 
necessary,  as  with  the  Abbe  camera  lucida  (£  182),  to  have  the  Wollaston  prism 
properly  arranged  with  reference  to  the  axis  of  the  microscope  and  the  eye-point. 
If  it  is  not,  one  will  be  unable  to  see  the  image  well,  and  may  be  entirely  unable 
to  see  the  pencil  and  the  image  at  the  same  time.  Again,  as  rays  from  the  micro- 
scope and  from  the  drawing  surface  must  enter  independent  parts  of  the  pupil  of 
the  same  eye,  one  must  hold  the  eye  so  that  the  pupil  is  partly  over  the  camera 
lucida  and  partly  over  the  drawing  surface.  One  can  tell  the  proper  position  by 
trial.  This  is  not  a  very  satisfactory  camera  to  draw7  with,  but  it  is  a  very  good 
form  to  measure  the  vertical  distance  of  250  rnm.  at  which  the  drawing  surface 
should  be  placed  when  determining  magnification  (\  162). 

§181.  *  Abbe  Camera  Lucida. — This  consists  of  a  cube  of  glass 
cut  into  two  triangular  prisms  and  silvered  on  the  cut  surface  of  the 


*For  some  persons  the  image  and  drawing  surface,  pencil,  etc.,  do  not  appear 
on  the  drawing  board  as  stated  above,  but  under  the  microscope,  according  to  the 
general  principle  that  "objects  appear  in  space  where  they  could  be  touched 
along  a  perpendicular  to  the  retinal  surface  stimulated," — that  is  in  the  line  of 
rays  entering  the  eye.  This  is  always  the  case  with  the  Wollaston  camera  lucida. 
The  explanation  of  the  apparent  location  of  the  image,  etc.,  on  the  drawing  board 
with  the  Abbe  camera  lucida  is  that  the  attention  is  concentrated  upon  the  draw- 
ing surface  rather  than  upon  the  object  under  the  microscope  (Dr.  W.  B. 
Pillsbury). 


CH.  F] 


DRA  WING   WITH  THE  MICROSCOPE 


125 


upper  one.  A  small  oval  hole  is  then  cut  out  of  the  center  of  the  sil- 
vered surface  and  the  two  prisms  are  cemented  together  in  the  form  of 
the  original  cube  with  a  perforated  45  degree  mirror  within  it  (Fig. 
109,  ab).  The  upper  surface  of  the  cube  is  covered  by  a  perforated 
metal  plate.  This  cube  is  placed  over  the  ocular  in  such  a  way  that 
the  light  from  the  microscope  passes  through  the  hole  in  the  silvered 
face  and  thence  directly  to  the  eye.  Light  from  the  drawing  surface 
is  reflected  by  a  mirror  to  the  silvered  surface  of  the  prism  and  reflect- 
ed by  this  surface  to  the  eye  in 
company  with  the  rays  from  the 
microscope,  so  that  the  two  fields 
appear  as  one,  and  the  image  is 
seen  as  if  on  the  drawing  surface 
(Figs.  109,  114).  It  is  designed 
for  use  with  a  vertical  micro- 
scope, but  see  §  184. 

FIG.  112.  Wollaston's  Camera 
Lucida,  showing  the  rays  from  the 
microscope  and  from,  the  drawing  sur- 
face, and  the  position  of  the  pupil  of 
the  eye. 

For  full  explanation  see  Fig.  97. 


§  182.  Arrangement  of  the  Camera  Lucida  Prism. — In  plac- 
ing this  camera  lucida  over  the  ocular  for  drawing  or  the  determination 
of  magnification,  the  center  of  the  hole  in  the  silvered  surface  is  placed 
in  the  optic  axis  of  the  microscope.  This  is  done  by  properly  arrang- 
ing the  centering  screws  that  clamp  the  camera  to  the  microscope  tube 
or  ocular.  The  perforation  in  the  silvered  surface  must  also  be  at  the 
level  of  the  eye-point.  In  other  words  the  prism  must  be  so  arranged 
vertically  and  horizontally  that  the  hole  in  the  silvered  surface  will  be 
in  the  axis  of  the  microscope  and  coincident  with  the  eye-point  of 
the  ocular.  If  it  is  above  or  below,  or  to  one  side  of  the  eye-point, 
part  or  all  of  the  field  of  the  microscope  will  be  cutoff.  As  stated 
above,  the  centering  screws  are  for  the  proper  horizontal  arrangement 
of  the  prism.  The  prism  is  set  at  the  right  height  by  the  makers  for 
the  eye-point  of  a  medium  ocular.  If  one  desires  to  use  an  ocular 
with  the  eye-point  farther  away  or  nearer,  as  in  using  high  or  low 
oculars  the  position  of  the  eye-point  may  be  determined  as  directd  in 


126  DRAWING  WITH  THE  MICROSCOPE  \_CH.   V 

§  59  and  the  prism  loosened  and  raised  or  lowered  to  the  proper  level  ; 
but  in  doing  this  one  should  avoid  setting  the  prism  obliquely  to  the 
mirror. 

In  the  latest  and  best  forms  of  this  camera  lucida  special  arrange- 
ments have  been  made  for  raising  or  lowering  the  prism  so  that  it  may 
be  used  with  equal  satisfaction  on  oculars  with  the  eye-point  at  differ- 
ent levels,  and  the  prism  is  hinged  to  turn  aside  without  disturbing 
the  mirror. 

One  can  determine  when   the 
camera  is  in  a   proper  position  by 
looking  into  the  microscope  through 
it.     If  the  field  of  the  microscope 
appears  as  a  circle  and  of  about  the 
same  size   as  without   the   camera 
lucida,  then  the  prism  is  in  a  proper 
FIG.  113.     One  of  the  latest  and      position.     If  one  side  of  the  field  is 
best  forms  of  the  Abbe  Camera  Lucida       d     fc      ^         h        rf          .          Qne  ^ 

(Bausch  &  Lomb  Optical  Co.). 

of  the  center  ;  if  the  field  is  consid- 
erably smaller  than  when  the  prism  is  turned  off  the  ocular,  it  indicates 
that  it  is  not  at  the  correct  level,  i.  e.,  it  is  above  or  below  the  eye- 
point. 

§  183.  Arrangement  of  the  Mirror  and  the  Drawing  Surface. 
—The  Abbe  camera  lucida  was  designed  for  use  with  a  vertical  micro- 
scope (Fig.  109).  On  a  vertical  microscope  if  the  mirror  is  set  at  an 
angle  of  45°,  the  axial  ray  will  be  at  right  angles  with  the  table  top  or  a 
drawing  board  which  is  horizontal,  and  a  drawing  made  under  these 
conditions  will  be  in  true  proportion  and  not  distorted.  The  stage  of 
most  microscopes,  however,  extends  out  so  far  at  the  sides  that  with 
a  45°  mirror  the  image  appears  in  part  on  the  stage  of  the  microscope. 
In  order  to  avoid  this  the  mirror  may  be  depressed  to  some  point  below 
45°,  say  at  40°  or  35° (Fig.  1 14).  But  as  the  axial  ray  from  the  mirror 
to  the  prism  must  still  be  reflected  horizontally,  it  follows  that  the  axial 
ray  will  no  longer  form  an  angle  of  90  degrees  with  the  drawing  sur- 
face, but  a  greater  angle.  If  the  mirror  is  depressed  to  35°,  then  the 
axial  ray  must  take  an  angle  of  110°  with  a  horizontal  drawing  surface 
(see  the  geometrical  figure  Fig.  114,  A).  To  make  the  angle  90°  again, 
so  that  there  shall  be  no  distortion,  the  drawing  board  must  be  raised 
toward  the  microscope  20°.  The  general  rule  is  to  raise  the  draw- 
ing board  twice  as  many  degrees  toward  the  microscope  as  the 
mirror  is  depressed  below  45°.  Practically  the  field  for  drawing 


CH.  F]  DRAWING   WITH  THE  MICROSCOPE 

B 


I27 


FIG.  114.  C 

Abbe  Camera  Lucida  in  position  to  avoid  distortion. 

FIG.  114. —  The  Abbe  Camera  Lucida  with  the  mirror  at  35°. 

Axis,  Axis.     Axial  ray  from  the  microscope  and  from  the  drawing  surface. 

A.  B.     Drawing  surface  raised  toward  the  microscope  20°. 

Foot.     The  foot  or  base  of  the  microscope. 

Mirror  with  quadrant  (Q).     The  mirror  is  seen  to  be  at  an  angle  of  35°. 

Ocular.     Ocular  of  the  Microscope. 

P.  P.    Drawing  pencil  and  the  cubical  prism  over  the  ocular. 

W.      Wedge  to  support  the  drawing  board. 

A.  Geometrical  figure  of  the  preceding,  showing  the  angles  made  by  the 
axial  ray  with  the  mirror  and  the  necessary  elevation  of  the  drawing  board  to 
avoid  distortion.     From  the  equality  of  opposite  angles,  the  angle  of  the  axial  ray 
reflected  at  jj°  must  make  an  angle  of  110°  with  a  horizontal  drawing  board.    The 
board  must  then  be  elevated  toward  the  microscope  20°  in  order  that  the  axial  ray 
may  be  perpendicular  to  it,  and  thus  fulfill  the  requirements  necessary  to  avoid 
distortion  ($  779,  183}. 

B.  Upper  view  of  the  prism  of  the  camera  lucid  a.     A  considerable  portion  of 
the  face  of  the  prism  is  covered,  and  the  opening  in  the  silvered  surface  appears 
oval. 

C.  Quadrant  to  be  attached  to  the  mirror  of  the  Abbe  Camera  Lucida  to  in- 
dicate the  angle  of  the  mirror.     As  the  angle  is  nearly  always  45° ,  40°,  or  55°, 
only  those  angles  are  shown. 


128  DRAWING   WITH  THE  MICROSCOPE  \CH.  V 

can  always  be  made  free  of  the  stage  of  the  microscope,  at  45°,  at  40°, 
or  at  35°.  In  the  first  case  (45°  mirror)  the  drawing  surface  should 
be  horizontal,  in  the  second  case  (40°  mirror)  the  drawing  surface 
should  be  elevated  10°,  and  in  the  third  case  (35°  mirror)  the  drawing 
board  should  be  elevated  20°  toward  the  microscope.  Furthermore  it 
is  necessary  in  using  an  elevated  drawing  board  to  have  the  mirror  bar 
project  directly  laterally  so  that  the  edges  of  the  mirror  will  be  in 
planes  parallel  with  the  edges  of  the  drawing  board,  otherwise  there 
will  be  front  to  back  distortion,  although  the  elevation  of  the  drawing 
board  would  avoid  right  to  left  distortion.  If  one  has  a  micrometer 
ruled  in  squares  (net  micrometer}  the  distortion  produced  by  not  having 
the  axial  ray  at  right  angles  with  the  drawing  surface  may  be  very 
strikingly  shown.  For  example,  set  the  mirror  at  35°  and  use  a  hori- 
zontal drawing  board.  With  a  pencil  make  dots  at  the  corners  of 
some  of  the  squares,  and  then  with  a  straight  edge  connect  the  dots. 
The  figures  will  be  considerably  longer  from  right  to  left  than  from 
front  to  back.  Circles  in  the  object  would  appear  as  ellipses  in  the 
drawings,  the  major  axis  being  from  right  to  left. 

The  angle  of  the  mirror  may  be  determined  with  a  protractor,  but 
that  is  troublesome.  It  is  much  more  satisfactory  to  have  a  quadrant 
attached  to  the  mirror  and  an  indicator  on  the  projecting  arm  of  the 
mirror.  If  the  quadrant  is  graduated  throughout  its  entire  extent,  or 
preferably  at  three  points,  45°,  40°  and  35°,  one  can  set  the  mirror  at  a 
known  angle  in  a  moment,  then  the  drawing  board  can  be  hinged  and 
the  elevation  of  10°  and  20°  determined  with  a  protractor.  The  draw- 
ing board  is  very  conveniently  held  up  by  a  broad  wedge.  By  marking 
the  position  of  the  wedge  for  10°  and  20°  the  protractor  need  be  used 
but  once,  then  the  wedge  may  be  put  into  position  at  any  time  for  the 
proper  elevation. 

§  184.  Abbe  Camera  and  Inclined  Microscope. — It  is  very 
fatiguing  to  draw  continuously  with  a  vertical  microscope,  and  many 
mounted  objects  admit  of  an  inclination  of  the  microscope,  when  one 
can  sit  and  work  in  a  more  comfortable  position.  The  Abbe  camera  is 
as  perfectly  adapted  to  use  with  an  inclined  as  with  a  vertical  micro- 
scope. All  that  is  requisite  is  to  be  sure  that  the  fundamental  law  is 
observed  regarding  the  axial  ray  of  the  image  and  the  drawing  surface, 
viz. ,  that  they  should  be  at  right  angles.  This  is  very  easily  accom- 
plished as  follows  :  The  drawing  board  is  raised  toward  the  microscope 
twice  as  many  degrees  as  the  mirror  is  depressed  below  45°  (§  183), 
then  it  is  raised  exactly  as  many  degrees  as  the  microscope  is  inclined, 


CH.  V\  DRAWING  WITH  THE  MICROSCOPE  129 

and  in  the  same  direction,  that  is,  so  the  end  of  the  drawing  board 
shall  be  in  a  plane  parallel  with  the  stage  of  the  microscope.  The 
mirror  must  have  its  edges  in  planes  parallel  with  the  edges  of  the 
drawing  board  also  (Figs.  115,  116.) 


FIG.     115.       Arrangement    of        jo" 
the  drawing  board  for  using  the 
microscope  in  an  inclined  position 
with  the  Abbe  camera  lucida  (de- 
signed by  Mrs.  S.  P.  Gage,  1887.) 


§  185.  Drawing  with  the  Abbe  Camera  Lucida. — (A)  The 
light  from  the  microscope  and  from  the  drawing  surface  should  be  of 
nearly  equal  intensity,  so  that  the  image  and  the  drawing  pencil  can  be 
seen  with  about  equal  distinctness.  This  may  be  accomplished  with 
very  low  powers  (16  mm.  and  lower  objectives)  by  covering  the  mirror 
of  the  microscope  with  white  paper  when  transparent  objects  are  to  be 
drawn.  For  high  powers  it  is  best  to  use  a  substage  condenser.  Often 
the  light  may  be  balanced  by  using  a  larger  or  smaller  opening  in  the 
diaphragm.  One  can  tell  which  field  is  excessively  illuminated,  for 
it  is  the  one  in  which  objects  are  most  distinctly  seen.  If  it  is  the 
microscopic,  then  the  image  of  the  microscopic  object  is  very  distinct 
and  the  pencil  is  invisible  or  very  indistinct.  If  the  drawing  surface  is 
too  brilliantly  lighted  the  pencil  can  be  seen  clearly,  but  the  micro- 
scopic image  will  be  obscure. 

When  opaque  objects,  that  is  objects  which  must  be  lighted  with 
reflected  light  (§  63),  like  dark  colored  insects,  etc.,  are  to  be  drawn 
the  light  must  usually  be  concentrated  upon  the  object  in  some  way. 
The  microscope  may  be  placed  in  a  very  strong  light  and  the  drawing 
board  shaded  or  the  light  may  be  concentrated  upon  the  object  by 
means  of  a  concave  mirror  or  a  bull's  eye  condenser  (Fig.  53). 

If  the  drawing  surface  is  too  brilliantly  illuminated,  it  may  be 
shaded  by  placing  a  book  or  a  ground  glass  screen  between  it  and  the 
window,  also  by  putting  one  or  more  smoked  glasses  in  the  path  of  the 
ra}7s  from  the  mirror  (Fig.  109  G).  If  the  light  in  the  microscope  is 
too  intense,  it  may  be  lessened  by  using  white  paper  over  the  mirror, 
or  by  a  ground  glass  screen  between  the  microscope  mirror  and  the 
source  of  light  (Piersol,  Amer.  M.  M.  Jour.,  1888,  p.  103).  It  is  also 
an  excellent  plan  to  blacken  the  end  of  the  drawing  pencil  with  carbon 


130 


DRAWING   WITH  THE  MICROSCOPE 


\CH.  V 


ink.  Sometimes  it  is  easier  to  draw  on  a  black  surface,  using  a  white 
pencil  or  style.  The  carbon  paper  used  in  manifolding  letters,  etc., 
may  be  used,  or  ordinary  black  paper  may  be  lightly  rubbed  on  one 
side  with  a  moderately  soft  lead  pencil.  Place  the  black  paper  over 
white  paper  and  trace  the  outlines  with  a  pointed  style  of  ivory  or 
bone.  A  corresponding  dark  line  will  appear  on  the  white  paper 
beneath.  (Jour.  Roy.  Micr.  Soc.,  1883,  p.  423). 


FIG.  1 1 6.  Bernhard's  Drawing  Board  for  the  Abbe  Camera  Lucida.  This 
drawing  board  is  adjustable  vertically,  and  the  board  may  be  inclined  to  prevent 
distortion.  It  is  also  arranged  for  use  with  an  inclined  microscope,  having  the 
base  board  hinged.  Microscope  and  drawing  surface  are  then  inclined  together. 
(Zeit.  wiss.  Mikroskopie,  vol.  vi  (1894,  p.  208).  (Zeiss  Catalog}. 

(A)  It  is  desirable  to  have  the  drawing  paper  fastened  with  thumb 
tacks,  or  in  some  other  way.  (B)  The  lines  made  while  using  the 
camera  lucida  should  be  very  light,  as  they  are  liable  to  be  irregular. 
(C)  Only  outlines  are  drawn  and  parts  located  with  a  camera  lucida. 
Details  are  put  in  free-hand.  (D)  It  is  sometimes  desirable  to  draw 
the  outline  of  an  object  with  a  moderate  power  and  add  the  details  with 
a  higher  power.  If  this  is  done  it  should  always  be  clearly  stated.  It 
is  advisable  to  do  this  only  with  objects  in  which  the  same  structure  is 
many  times  duplicated,  as  a  nerve  or  a  muscle.  In  such  an  object  all 


CH.  F]  DRAWING   WITH  THE  MICROSCOPE  131 

the  different  structures  could  be  shown,  and  by  omitting  some  of  the 
fibers  the  others  could  be  made  plainer  without  an  undesirable  enlarge- 
ment of  the  entire  figure. 

(E)  If  a  drawing  of  a  given  size  is  desired  and  it  cannot  be  ob- 
tained by  any  combination  of  oculars,  objectives  and  lengths  of  the 
tube  of  the  microscope,  the  distance  between  the  camera  lucida  and 
the  table  may  be  increased  or  diminished  until  the  image  is  of  the 
desired  size.     This  distance  is  easily  changed  by  the  use  of  a  book  or 
a  block,  but  more  conveniently  if  one  has  a  drawing  board  with  adjust- 
able drawing  surface  like  that  shown  in  Fig.  1 16.     The  image  of  a  few 
spaces  of  the  micrometer  will  give  scale  of  enlargement,  or  the  power 
may  be  determined  for  the  special  case  (§  186-187). 

(F)  It  is  of  the  greatest  advantage,  as  suggested  by  Heinsius  (Zeit. 
w.  Mikr.,  1889,  P-  367)  i  to  have  the  camera  lucida  hinged  so  that  the 
prism  may  be  turned  off  the  ocular  for  a  moment's  glance  at  the  prepa- 
ration, and  then  returned  in  place  without  the  necessity  of  loosening 
screws  and  readjusting  the  camera.     This  form  is  now  made  by  several 
opticians,  and  the  quadrant  is  added  by  some.   (Fig.  114.)  -Any  skilled 
mechanic  can  add  the  quadrant. 

§  186.  Magnification  of  the  Microscope  and  size  of  Draw- 
ings with  the  Abbe  Camera  Lucida. — In  determining  the  standard 
distance  of  250  millimeters  at  which  to  measure  the  image  in  getting 
the  magnification  of  the  microscope,  it  is  necessary  to  measure  from 
the  point  marked  P  on  the  prism  (Fig.  109)  to  the  axis  of  the  mirror 
and  then  vertically  to  the  drawing  board. 

In  getting  the  scale  to  which  a  drawing  is  enlarged  the  best  way 
is  to  remove  the  preparation  and  put  in  its  place  a  stage  micrometer, 
and  to  trace  a  few  (5  or  10)  of  its  lines  upon  one  corner  of  the  drawing. 
The  value  of  the  spaces  of  the  micrometer  being  given,  thus  : 


,^th  mm. 

FIG.  117.     Showing  the  method  of  indicating  the  scale  at  which  a  drawing 
was  made. 

The  enlargement  of  the  figure  can  then  be  accurately  determined 
at  any  time  by  measuring  with  a  steel  scale  the  length  of  the  image  of 
the  micrometer  spaces  and  dividing  it  by  their  known  size. 

Thus,  suppose  the  5  spaces  of  the  scale  of  enlargement  given  with 
a  drawing  were  found  to  measure  25  millimeters  and  the  spaces  on  the 


132  DRAWING  WITH  THE  MICROSCOPE  [CH.  V 

micrometer  were  y1  th  millimeter,  then  the  enlargement  would  be 
25  -3-  yf^  =  500.  That  is,  the  image  was  drawn  at  a  magnification  of 
500  diameters. 

If  the  micrometer  scale  is  added  to  every  drawing,  there  is  no 
need  of  troubling  one's  self  about  the  exact  distance  at  which  the 
drawing  is  made,  convenience  may  settle  that,  as  the  special  magnifi- 
cation in  each  case  may  be  determined  from  the  scale  accompanying 
the  picture.  It  should  be  remembered,  however,  that  the  conditions 
when  the  scale  is  drawn  must  be  exactly  as  when  the  drawing  was 
made. 

§  187.  Drawing  at  Slight  Magnification. — Some  objects  are  of 
considerable  size  and  for  drawings  should  be  enlarged  but  a  few  diame- 
ters,— 5  to  20.  By  using  sufficiently  low  objectives  and  different  ocu- 
lars a  great  range  may  be  obtained.  Frequently,  however,  the  range 
must  be  still  further  increased.  For  a  moderate  increase  in  size  the 
drawing  surface  may  be  put  farther  off,  or,  as  one  more  commonly 
needs  less  rather  than  greater  magnification,  the  drawing  surface  may 
be  brought  nearer  the  mirror  of  the  camera  lucida  by  piling  books  or 
other  objects  on  the  drawing  board.  If  one  takes  the  precaution  to 
draw  a  scale  on  the  figure  under  the  same  conditions,  its  enlargement 
can  be  readily  determined  (§  186). 

A  very  satisfactory  way  to  draw  at  low  magnifications  is  to  use  a 
simple  microscope  and  arrange  a  camera  lucida  over  it  as  over  the  ocular. 
In  this  way  one  may  get  drawings  at  almost  any  low  magnification. 

If  one  has  many 
large  objects  to  draw  at  a 
low  magnification,  then 
some  form  of  embryo- 
graph  is  very  conven- 
ient. (Jour.  Roy.  Micr., 
Soc.,  1899,  P-  223.)  The 
writer  has  made  use  of 
a  photographic  camera 
and  different  photo- 
graphic objectives  for  the 
purpose.  The  object  is 

FIG.  1 1 8.     Camera   lucida  for  drawing  objects   natural  size.     (H.   Bausch 
Jour.  Applied  Microscopy,  vol.  Hi  (/£<%>,  p.  801). 

illuminated  as  if  for  a  photograph  and  in  place  of  the  ground  glass  a 
plain  glass  is  used  and  on  this  some  tracing  paper  is  stretched.  Noth- 


CH.  F] 


DRA  WING   WITH  THE  MICROSCOPE 


133 


ing  is  then  easier  than  to  trace  the  outlines  of  the  object.    See  also  Ch. 
VIII. 

REFERENCES 

Beale,  31,  355  ;  Behrens,  Kossel  and  Schiefferdecker,  77  ;  Carpenter-Dallinger, 
278  ;  Van  Heurck,  91  ;  American  Naturalist,  1886,  p.  1071,  1887,  pp.  1040-1043; 
Amer.  Monthly  Micr.  Jour.,  1888,  p.  103,  1890,  p.  94  ;  Jour.  Roy.  Micr.  Soc.,  iSSi, 
p.  819,  1882,  p.  402,  1883,  pp.  283,  560,  1884,  p.  115,  1886,  p.  516,  1888,  pp.  113,  809, 
798;  Zeit.  wiss.  Mikroskopie,  1884,  pp.  1-21,  1889,  p.  367,  1893,  pp.  289-295. 
Here  is  described  an  excellent  apparatus  made  by  Winkel.  Consult  also  the  latest 
catalogs  of  the  opticians. 


1O    CENTIMETER    RULE 

The  upper  edge  is  in  millimeters,  the  lower  in  centimeters,  and  half  centimeters. 


THE    METRIC    SYSTEM 


UNITS.  The  most  commonly  used  divisions  and  multiples. 

.„  (Centimeter  (c.m.),  i-iooth  Meter  ;   Millimeter  (m.m.),  i-ioooth  Meter:   Micron 
iR<          (,x  ).  i-ioooth  Millimeter  ;  the  Micron  is  the  unit  in  Micrometry  &  166). 


LENGTH. 


(  Kilometer,  1000  Meters  ;  used  in  measuring  roads  and  other  long  distances. 


THE    GRAM    FOR  \  Milligram  (m.g.),  i-ioooth  Gram. 
WEIGHT.    .     .      >  Kilogram,  1000  Grams,  used  for  ordinary  masses,  like  groceries,  etc. 

THE    LITER    FOR  i  Cubic  Centimeter  (c.c. ),  i-ioooth  Liter.    This  is  more  common  than  the  correct 
CAPACITY.      .     \         form,  Milliliter. 

Divisions  of  the  Units  are  indicated  by  the  Latin  prefixes  :  deci,  i-ioth  ;  centi,  i-iooth  ;  Milli, 
i-ioooth  ;  Micro,  i-i,ooo,oooth  of  any  unit. 

Multiples  are  designated  by  Greek  prefixes  :  deka,  10  times  ;  hecto,  100  times  ;  kilo^  1000  times 
myria,  10,000  times  ;  Mega,  1,000,000  times  any  unit. 


CHAPTER  VI 

MICRO-SPECTROSCOPE   AND   POLARISCOPE, 

MICRO-CHEMISTRY,     MICRO-MATALLOGRAPHY, 

TEXTILE   FIBERS 


APPARATUS   AND    MATERIAL    REQUIRED    FOR    THIS    CHAPTER 

Compound  microscope  ;  Micro-spectroscope  (g  188)  ;  Watch-glasses  and  small 
vials,  slides  and  covers  ($  207)  ;  Various  substances  for  examination  (as  blood  and 
ammonium  sulphide,  permanganate  of  potash,  chlorophyll,  some  colored  fruit, 
etc.,  (§  208-217);  Micro-polarizer  (§  218);  Selenite  plate  (§  227);  Various  doubly 
refracting  objects,  as  crystals,  textile  fibers,  starch,  section  of  bone  ;  Various 
chemicals,  metals,  etc. 

MICRO-SPECTROSCOPE 

\  188.  A  Micro-Spectroscope,  Spectroscopic  or  Spectral  Ocular,  is  a  direct 
vision  spectroscope  in  connection  with  a  microscope  ocular.  The  one  devised  by 
Abbe  and  made  by  Zeiss  consists  of  a  direct  vision  spectroscope  prism  of  the  Amici 
pattern,  and  of  considerable  dispersion,  placed  over  the  ocular  of  the  microscope. 
This  direct  vision  or  Amici  prism  consists  of  a  single  triangular  prism  of  heavy 
flint  glass  in  the'  middle  and  one  of  crown  glass  on  each  side,  the  edge  of  the 
crown  glass  prisms  pointing  toward  the  base  of  the  flint  glass  prism,  i.  e.,  the  edge 
of  the  crown  and  flint  glass  prisms  point  in  opposite  directions.  The  flint  glass 
prism  serves  to  give  the  dispersion  or  separation  into  colors,  while  the  crown  glass 
prisms  serve  to  make  the  emergent  rays  approximately  parallel  with  the  incident 
rays,  so  that  one  looks  directly  into  the  prism  along  the  axis  of  the  microscope. 

The  Amici  prism  is  in  a  special  tube  which  is  hinged  to  the  ocular  and  held  in 
position  by  a  spring.  It  may  be  swung  free  of  the  ocular.  In  connection  with 
the  ocular  is  the  slit  mechanism  and  a  prism  for  reflecting  horizontal  rays  verti- 
cally for  the  purpose  of  obtaining  a  comparison  spectrum  ($  201).  Finally  near 
the  top  is  a  lateral  tube  with  mirror  for  the  purpose  of  projecting  an  Angstrom 
scale  of  wave  lengths  upon  the  spectrum  ($  202,  Figs.  119-120.) 

\  189.  Apparent  Reversal  of  the  Position  of  the  Colors  in  a  Direct  Vision 
Spectroscope. — In  accordance  with  the  statements  in  §  188  the  dispersion  or  sepa- 
ration into  colors  is  given  by  the  flint  glass  prism  or  prisms  and  in  accordance 
with  the  general  law  that  the  waves  of  shortest  length,  blue,  etc.,  will  be  bent 
most,  the  colors  have  the  position  indicated  in  the  top  of  Fig.  123,  also  above 
Fig.  119.  But  if  one  looks  into  the  direct  vision  spectroscope  or  holds  the  eye 
close  to  the  single  prism  (Fig.  124),  the  colors  will  appear  reversed  as  if  the  red 
were  more  bent.  The  explanation  of  this  is  shown  in  Fig.  124,  where  it  can  be 


CH.  VI]        MICRO-SPECTROSCOPE  AND  POLARISCOPE 


135 


readily  seen  that  if  the  eye  is  placed  at  E,  close  to  the  prism,  the  different  colored 
rays  appear  in  the  direction  from  which  they  reach  the  eye  and  consequently 
are  crossed  in  being  projected  into  the  field  of  vision  and  the  real  position  is  in- 
verted. The  same  is  true  in  looking  into  the  micro-spectroscope.  The  actual 


Abbe's  Micro-spectroscope. 


FIG.  120. 

Slit  Mechanism  separately. 
(Plan  view,  Full  size.} 


FIG.  119. 

Longitudinal  Section  of 
the  whole  instrument. 
( yz  Full  size. ) 

"The  eye  lens  is  adjustable  so  as  to  accurately  focus  on  the  slit  situated  between 
the  lenses.  The  mechanism  for  contracting  and  expanding  the  slit  is  actuated  by 
the  screw  F and  causes  the  laminae  to  move  symmetrically  {Merz^s  movement). 
The  slit  may  be  made  sufficiently  wide  so  as  to  include  the  whole  visual  field.  The 
screw  H  serves  to  limit  the  length  of  the  slit  so  as  to  completely  fill  the  latter  with 
the  image  of  the  object  under  investigation  when  the  comparison  prism  is  inserted. 
The  comparison  prism  is  provided  with  a  lateral  frame  and  clips  to  hold  the  object 
and  the  illuminating  mirror.  All  these  parts  are  encased  in  a  drum  on  the  ocular. 

Above  the  eye-piece  is  placed  an  Amid  prism  of  great  dispersion  which  may  be 
turned  aside  about  the  pivot  K,  so  as  to  allow  of  the  adjustment  of  the  object.  The 
prism  is  retained  in  its  axial  position  by  the  spring  catch  L  A  scale  is  projected 
on  the  spectrum  by  means  of  a  scale  tube  and  mirror  attached  to  the  prism  casing. 
The  divisions  of  the  scale  indicate  in  decimals  of  a  micron  the  wave  length  of  the 
respective  section  of  the  spectrum.  The  screw  P  serves  to  adjust  the  scale  relative 
to  the  spectrum. 

The  instrument  is  inserted  in  the  tube  in  place  of  the  ordinary  eye-piece  and  is 
clamped  to  the  former  by  means  of  the  screw  M  in  such  a  position  that  the  mirrors 
A  and  O,  which  respectively  serve  to  illuminate  the  comparison  prism  and  the 
scale  of  wave  lengths  are  simultaneously  illuminated.'1'1  (Cut  loaned  by  Wm. 
Krafft,  N.  Y. ) 


136 


MICRO-SPECTROSCOPE  AND  POLARISCOPE        \_CH.  VI 


position  of  the  different  colors  may  be  determined  by  placing  some  ground  glass 
or  some  of  the  lens-paper  near  the  prism  and  observing  with  the  eye  at  the 
distance  of  distinct  vision.* 


j?o'Ur 

$(uctr 


FIG.  121.  Various  Spedrums. — All  except  that  of  sodium  were  obtained  by 
diffused  day-light  with  the  slit  of  such  a  width  as  gave  the  most  distinct  Fraunhofer 
lines. 

It  frequently  occurs^that  with  a  substance  giving  several  absorption  bands  (e. 
g.,  chlorophyll]  the  density  or  thickness  of  the  solution  must  be  varied  to  show  all 
the  different  bands  clearly. 

Solar  Spectrum. —  With  diffused  day-light  and  a  narrow  slit  the  spectrum  is  not 
visible  much  beyond  the  fixed  line  B.  In  order  to  extend  the  visible  spectrum  in 
the  red  to  the  line  A,  one  should  use  direct  sunlight  and  a  piece  of  ruby  glass  in 
place  of  the  watch-glass  in  Fig.  123. 

Sodium  Spectrum. —  The  line  spectrum  (§/?/)  of  sodium  obtained  by  lighting 
the  microscope  with  an  alcohol  flame  in  which  some  salt  of  sodium  is  glowing. 
With  the  micro-spectroscope  the  sodium  line  seen  in  the  solar  spectrum  and  with 
the  incandescent  sodium  appears  single,  except  under  very  favorable  circumstances. 
(|  102).  By  using  a  comparison  spectrum  of  day-light  with  the  sodium  spectrum 
the  light  and  dark  D-lines  will  be  seen  to  be  continuous  as  here  shown. 

Permanganate  of  Potash. — This  spectrum  is  characterized  by  the  presence  of 
five  absorption  bands  in  the  middle  of  the  spectrum  and  is  best  shown  by  using  a  ^ 
per  cent,  solution  of  permanganate  in  water  in  a  watch-glass  as  in  Fig.  123. 

Met-hemoglobin. —  The  absorption  spectrum  of  met-hemoglobin  is  characterized 
by  a  considerable  darkening  of  the  blue  end  of  the  spectrum  and  of  four  absorption 
bands,  one  in  the  red  near  the  line  C  and  two  between  D  and  E  nearly  in  the  place 
of  the  two  bands  of  oxy-hemoglobin  ;  finally  there  is  a  somewhat  faint,  wide,  band 
near-  F.  Such  a  met-hemoglobin  spectrum  is  best  obtained  by  making  a  solution  of 
blood  in  water  of  such  a  concentration  that  the  two  oxy-hemoglobin  bands  run 
together  (§  211],  and  then  adding  three  or  four  drops  of  a  ^  per  cent,  aqueous 
solution  of  permanganate  of  potash  or  a  few  drops  of  hydrogen  dioxid  (H2O2 ). 
Soon  the  bright  red  will  change  to  a  brownish  color,  when  it  may  be  examined. 

*The  author  wishes  to  acknowledge  the  aid  rendered  by  Professor  E.  L. 
Nichols  in  giving  the  explanation  offered  in  this  section. 


CH.  VI]         MICRO-SPECTROSCOPE  AND  POLARISCOPE  137 

VARIOUS   KINDS   OF  SPECTRA 

By  a  spectrum  is  meant  the  colored  bands  appearing  when  the  light  traverses 
a  dispersing  prism  or  a  diffraction  grating,  or  is  affected  in  any  way  to  separate 
the  different  wave  lengths  of  light  into  groups.  When  daylight  or  some  good 
artificial  light  is  thus  dispersed  one  gets  the  appearance  so  familiar  in  the 
rainbow. 

\  190.  Continuous  Spectrum. — In  case  a  good  artificial  light  as  the  electric 
light  is  used  the  various  rainbow  or  spectral  colors  merge  gradually  into  one 
another  in  passing  from  end  to  end  of  the  spectrum.  There  are  no  breaks  or  gaps. 

§  191.  Line  Spectrum. — If  a  gas  is  made  incandescent,  the  spectrum  it  pro- 
duces consists,  not  of  the  various  rainbow  colors,  but  of  sharp,  narrow,  bright  lines, 
the  color  depending  on  the  substance.  All  the  rest  of  the  spectrum  is  dark. 
These  line  spectra  are  very  strikingly  shown  by  various  metals  heated  till  they  are 
in  the  form  of  incandescent  vapor. 

\  192.  Absorption  Spectrum. — By  this  is  meant  a  spectrum  in  which  there  are 
dark  lines  or  bands  in  the  spectrum.  The  most  striking  and  interesting  of  the 
absorption  spectra  is  the  Solar  Spectrum ,  or  spectrum  of  sunlight.  If  this  is  exam- 
ined carefully  it  will  be  found  to  be  crossed  by  dark  lines,  the  appearance  being  as 
if  one  were  to  draw  pen  marks  across  a  continuous  spectrum  at  various  levels, 
sometimes  apparently  between  the  colors  and  sometimes  in  the  midst  of  a  color. 
These  dark  lines  are  the  so-called  Fraunhofer  Lines.  Some  of  the  principal  ones 
have  been  lettered  with  Roman  capitals,  A,  B,  C,  D,  E,  F,  G,  H,  commencing  at 
the  red  end.  The  meaning  of  these  lines  was  for  a  long  time  enigmatical,  but  it 
is  now  known  that  they  correspond  with  the  bright  lines  of  a  line  spectrum  (§  191). 
For  example,  if  sodium  is  put  in  the  flame  of  a  spirit  lamp  it  will  vaporize  and 
become  luminous.  If  this  light  is  examined  there  will  be  seen  one  or  two  bright 
yellow  bands  corresponding  in  position  with  D  of  the  solar  spectrum  (Fig.  121). 
If  now  the  spirit-lamp  flame,  colored  by  the  incandescent  sodium,  is  placed  in  the 
path  of  the  electric  light,  and  it  is  examined  as  before,  there  will  be  a  continuous 
spectrum,  except  for  dark  lines  in  place  of  the  bright  sodium  lines.  That  is,  the 
comparatively  cool  yellow  light  of  the  spirit  lamp  cuts  off  or  absorbs  the  intensely 
hot  yellow  light  of  the  electric  light ;  and  although  the  spirit  flame  sends  a  yellow 
light  to  the  spectroscope  it  is  so  faint  in  comparison  with  the  electric  light  that  the 
sodium  lines  appear  dark.  It  is  believed  that  in  the  sun's  atmosphere  there  are 
incandescent  metal  vapors  (sodium,  iron,  etc.),  but  that  they  are  so  cool  in  com- 
parison with  the  rays  of  their  wave  length  in  the  sun  that  the  cooler  light  of  the 
incandescent  metallic  vapors  absorb  the  light  of  corresponding  wave  length,  and 
are,  like  the  spirit  lamp-flame,  unable  to  make  up  the  loss,  and  therefore  the  pres- 
ence of  the  dark  lines. 

\  193.  Absorption  Spectra  from  Colored  Substances. — While  the  solar  spec- 
trum is  an  absorption  spectrum,  the  term  is  more  commonly  applied  to  the  spectra 
obtained  with  light  which  has  passed  through  or  has  been  reflected  from  colored 
objects  which  are  not  self-luminous. 

It  is  the  special  purpose  of  the  micro-spectroscope  to  investigate  the  spectra  of 
colored  objects  which  are  not  self-luminous,  i.  e.,  blood  and  other  liquids,  various 
minerals,  as  monazite,  etc.  The  spectra  obtained  by  examining  the  light  reflected 
from  these  colored  bodies  or  transmitted  through  them,  possess,  like  the  solar 


138 


MICRO-SPECTROSCOPE  AND  POLARISCOPE        \_CH.  VI 


spectrum  dark  lines  or  bands,  but  the  bands  are  usually  much  wider  and  less 
sharply  defined.  Their  number  and  position  depend  on  the  substance  or  its  con- 
stitution (Fig.  122),  and  their  width,  in  part,  upon  the  thickness  of  the  body. 
With  some  colored  bodies,  no  definite  bands  are  present.  The  spectrum  is  simply 
restricted  at  one  or  both  ends  and  various  of  the  other  colors  are  considerably 
lessened  in  intensity.  This  is  true  of  many  colored  fruits. 

\  194.     Angstrom   and    Stokes'  Law   of  Absorption    Spectra. — The  waves  of 
light  absorbed  by  a  body  when  light  is  transmitted  through  some  of  its  substance 


FIG.  122.  Absorption  spectrum  of  Oxy-hemoglobin  or  arterial  blood  (/)  and 
of  Ho  moglobin  or  venous  blood  (2).  (From  Gamgee  and  McMunn}. 

A,  B,  C,  D,  E,  F,  G,  H.  Some  of  the  Prinipal  Fraunhofer  lines  of  the  solar 
spectrum  (\  192}. 

.90,  .80,  .70,  .60,  .'jo,  .40.  Wave  lengths  in  microns,  as  shown  in  Angstrom's 
scale  (§  202).  It  will  be  seen  that  the  wave  lengths  increase  toward  the  red  and 
decrease  toward  the  violet  end  of  the  spectrmn. 

Red,  Yellow,  Orange,  etc.  Color  regions  of  the  spectrum.  Indigo  should 
come  between  the  blue  and  the  violet  to  complete  the  seven  colors  usually  given.  It 
was  omitted  through  inadvertence. 

are  precisely  the  waves  radiated  from  it  when  it  becomes  self-luminous.  For  ex- 
ample, a  piece  of  glass  that  is  yellow  when  cool,  gives  out  blue  light  when  it  is  hot 
enough  to  be  self-luminous.  Sodium  vapor  absorbs  two  bands  of  yellow  light  (D 
lines);  but  when  light  is  not  sent  through  it,  but  itself  is  luminous  and  examined 
as  a  source  of  light  its  spectrum  gives  bright  sodium  lines,  all  the  rest  of  the 
spectrum  being  dark  (Fig.  121). 

§  195.  Law  of  Color. — The  light  reaching  the  eye  from  a  colored,  solid, 
liquid  or  gaseous  body  lighted  with  white  light,  will  be  that  due  to  white  light  less 
the  light  waves  that  have  been  absorbed  by  the  colored  body.  Or  in  other  words, 
it  will  be  due  to  the  wave  lengths  of  light  that  finally  reach  the  eye  from  the  ob- 
ject. For  example,  a  thin  layer  of  blood  under  the  microscope  will  appear 
yellowish  green,  but  a  thick  layer  will  appear  pure  red.  If  now  these  two  layers 
are  examined  with  a  micro-spectroscope,  the  thin  layer  will  show  all  the  colors,  but 
the  red  end  will  be  slightly,  and  the  blue  end  considerably  restricted,  and  some  of 
the  colors  will  appear  considerably  lessened  in  intensity.  Finally  there  may  ap- 
pear two  shadow-like  bands,  or  if  the  layer  is  thick  enough,  two  well-defined 
dark  bands  in  the  green  (g  210). 

If  the  thick  layer  is  examined  in  the  same  way,  the  spectrum  will  show  only 
red  with  a  little  orange  light,  all  the  rest  being  absorbed.  Thus  the  spectroscope 
shows  which  colors  remain,  in  part  or  wholly,  and  it  is  the  mixture  of  this  remain- 
ing or  unabsorbed  light  that  gives  color  to  the  object. 


CH,  VI}        MICRO-SPECTROSCOPE  AND  POLARISCOTE  139 

\  196.  Complementary  Spectra. — While  it  is  believed  that  Angstrom's  law 
(I  194)  is  correct,  there  are  many  bodies  on  which  it  cannot  be  tested,  as  they 
change  in  chemical  or  molecular  constitution  before  reaching  a  sufficiently  high 
temperature  to  become  luminous.  There  are  compounds,  however,  like  those  of 
didymium,  erbium  and  terbium,  which  do  not  change  with  the  heat  necessary  to 
render  them  luminous,  and  with  them  the  incandescence  and  absorption  spectra 
are  mutually  complementary,  the  one  presenting  bright  lines  where  the  other 
presents  dark  ones  (Daniell). 

ADJUSTING   THE   MICRO-SPECTROSCOPE 

§  197.  The  micro-spectroscope,  or  spectroscopic  ocular,  is  put  in 
the  place  of  the  ordinary  ocular  in  the  microscope,  and  clamped  to  the 
top  of  the  tube  by  means  of  a  screw  for  the  purpose. 

§  198.  Adjustment  of  the  Slit. — In  place  of  the  ordinary  dia- 
phragm with  circular  opening,  the  spectral  ocular  has  a  diaphragm 
composed  of  two  movable  knife  edges  by  which  a  slit-like  opening  of 
greater  or  less  width  and  length  may  be  obtained  at  will  by  the  use  of 
screws  for  the  purpose.  To  adjust  the  slit,  depress  the  lever  holding 
the  prism-tube  in  position  over  the  ocular,  and  swing  the  prism  aside. 
One  can  then  look  into  the  ocular.  The  lateral  screw  should  be  used 
and  the  knife  edges  approached  till  they  appear  about  half  a  millimeter 
apart.  If  now  the  Amici  prism  is  put  back  in  place  and  the  micro- 
scope well  lighted,  one  will  see  a  spectrum  by  looking  into  the  upper 
end  of  the  spectroscope.  If  the  slit  is  too  wide,  the  colors  will  overlap 
in  the  middle  of  the  spectrum  and  be  pure  only  at  the  red  and  blue 
ends  ;  and  the  Fraunhofer  or  other  bands  in  the  spectrum  will  be 
faint  or  invisible.  Dust  on  the  edges  of  the  slit  gives  the  appearance 
of  longitudinal  streaks  on  the  spectrum. 

§  199.  Mutual  Arrangement  of  Slit  and  Prism. — In  order 
that  the  spectrum  may  appear  as  if  made  up  of  colored  bands  going 
directly  across  the  long  axis  of  the  spectrum,  the  slit  must  be  parallel 
with  the  refracting  edge  of  the  prism.  If  the  slit  and  prism  are  not 
thus  mutually  arranged,  the  colored  bands  will  appear  oblique,  and 
the  whole  spectrum  may  be  greatly  narrowed.  If  the  colored  bands 
are  oblique,  grasp  the  prism  tube  and  slowly  rotate  it  to  the  right  or  to 
the  left  until  the  various  colored  bands  extend  directly  across  the  spec- 
trum. 

§  200.  Focusing  the  Slit. — In  order  that  the  lines  or  bands  in 
the  spectrum  shall  be  sharply  denned,  the  eye-lens  of  the  ocular  should 
be  accurately  focused  on  the  slit.  The  eye-lens  is  movable,  and  when 
the  prism  is  swung  aside  it  is  very  easy  to  focus  the  slit  as  one  focused 


140 


MICRO-SPECTROSCOPE  AND  POLARISCOPE        \_CH.  VI 


for  the  ocular  micrometer  (§  172).  If  one  now  uses  daylight  there 
will  be  seen  in  the  spectrum  the  dark  Fraunhofer  lines  (Fig.  121  E.  F., 
etc.). 


Coxrx]}. 

••  ' 
*•*! 

\\.xi?trt 

\ 

i   i  '. 

i  C    "i   ' 

/ 

FIG.  123.  FIG.  124.  FIG.  125. 

FIG.  123.  (i).  Section  of  the  tube  and  stage  of  the  microscope  with  the  spectral 
ocular  or  micro-spectroscope  in  position. 

Amid  Prism  (\  188). —  The  direct  vision  prism  of  Amid  in  which  the  central 
shaded  pi  ism  of  flint  glass  gives  the  dispersion  or  separation  into  colors,  luhile  the 


CH.   VI]        MICRO-SPECTROSCOPE  AND  POLARISCOPE  141 

end  prisms  of  crown  glass  cause  the  rays  to  emerge  approximately  parallel  with 
the  axis  of  the  microscope.  A  single  ray  is  represented  as  entering  the  prism  and 
this  is  divided  into  three  groups  (Red,  Yellow,  Blue},  which  emerge  from  the 
prism,  the  red  being  least  and  the  blue  most  bent  toward  the  base  of  the  flint  prism 
(see  Fig.  124). 

Hinge. —  The  hinge  on  which  the  prism  tube  turns  when  it  is  swung  off  the 
ocular. 

Ocular  (\  1 88) — The  ocular  in  which  the  slit  mechanism  takes  the  place  of  the 
diaphragm  (§198).  The  eye-lens  is  movable  as  in  a  micrometer  ocular,  so  that 
the  slit  may  be  accurately  focused  for  the  different  colors  (|  200). 

S.     Screw  for  setting  the  scale  of  wave  lengths  ( \  202) . 

S' ' .     Screw  for  regulating  the  width  of  the  slit  (  \  198). 

S//.     Screw  for  clamping  the  micro-spectroscope  to  the  tube  of  the  microscope. 

Scale  Tube. —  The  tube  near  the  upper  end  containing  the  Angstrom  scale  and 
the  lenses  for  projecting  the  image  upon  the  upper  face  of  the  Amid  prism,  whence 
it  is  reflected  upward  to  the  eye  with  the  different  colored  rays.  At  the  right  is  a 
special  mirror  for  lighting  the  scale. 

Slit. —  The  linear  opening  between  the  knife  edges.  Through  the  slit  the  light 
passes  to  the  prism.  It  must  be  arranged  parallel  with  the  refracting  edge  of  the 
prism,  and  of  such  a  width  that  the  Fraunhofer  or  Fixed  Lines  are  very  clearly  and 
sharply  defined  when  the  eye-lens  is  properly  Jocused  (\  198-200). 

Stage. — The  stage  of  the  microscope.  This  supports  a  watch-glass  with  sloping 
sides  for  containing  the  colored  liquid  to  be  examined. 

(3)  Comparison  Prism  with  tube  for  colored  liquid  (C.  L.},  and  mirror.    The 
prism  reflects  horizontal  rays  vertically,  so  that  when  the  prism  is  made  to  cover 
part  of  the  slit  two  parallel  spectra  may  be  seen,  one  from  light  sent  directly  through 
the  entire  microscope  and  one  from  the  light  reflected  upward  from  the  comparison 
prism. 

(4)  View  of  the  Slit  Mechanism  from  below. — Slit,  the  linear  space  between 
the  knife  edges  through  which  the  light  passes. 

P.     Comparison  prism  beneath  the  slit  and  covering  part  of  it  at  will. 

S.  S' '.     Screws  for  regulating  the  length  and  width  of  the  slit. 

FIG.  124.  Flint-Glass  Prism  showing  the  separation  or  dispersion  of  white 
light  into  the  three  groups  of  colored  rays  (Red,  Yellow,  Blue],  the  blue  rays  being 
bent  the  most  from  the  refracting  edge  (\  189). 

FIG.  125.  Sectional  View  of  a  Microscope  with  the  Polariscope  in  position 
(8218-227). 

Analyzer  and  Polarizer. — They  are  represented  with  corresponding  faces  par- 
allel so  that  the  polarized  beam  could  traverse  freely  the  analyzer.  If  either  Nicol 
were  rotated  90°  they  would  be  crossed  and  no  light  would  traverse  the  analyzer 
unless  some  polarizing  substance  were  used  as  object,  (a)  Slot  in  the  analyzer 
tube  so  that  the  analyzer  may  be  raised  or  lowered  to  adjust  it  for  difference  of 
level  of  the  eye- point  in  different  oculars  (\  59,  200). 

Pointer  and  Scale. —  The  pointer  attached  to  the  analyzer  and  the  scale  or  divided 
circle  clamped  (by  the  screw  S)  to  the  tube  of  the  microscope.  The  pointer  and 
scale  enable  one  to  determine  the  exact  amount  of  rotation  of  the  analyzer  (\  220). 

Object — The  object  whose  character  is  to  be  investigated  by  polarized  light. 


142  MICRO-SPECTROSCOPE  AND  POLAR/SCOPE        [CH.  VI 

To  show  the  necessity  of  focusing  the  slit,  move  the  eye-lens  down 
or  up  as  far  as  possible,  and  the  Fraunhofer  lines  cannot  be  seen. 
While  looking  into  the  spectroscope  move  the  ocular  lens  up  or  down, 
and  when  it  is  focused  the  Fraunhofer  lines  will  reappear.  As  the  dif- 
ferent colors  of  the  spectrum  have  different  wave  lengths,  it  is  neces- 
sary to  focus  the  slit  for  each  color  if  the  sharpest  possible  pictures  are 
desired. 

It  will  be  found  that  the  eye-lens  of  the  ocular  must  be  farther 
from  the  slit  for  the  sharpest  focus  of  the  red  end  than  for  the  sharpest 
focus  of  the  lines  at  the  blue  end.  This  is  because  the  wave  length  of 
red  is  markedly  greater  than  for  blue  light. 

Longitudinal  dark  lines  of  the  spectrum  may  be  due  to  irregular- 
ity of  the  edge  of  the  slit  or  to  the  presence  of  dust.  They  are  most 
troublesome  with  a  very  narrow  slit. 

§  201.  Comparison  or  Double  Spectrum. — In  order  to  com- 
pare the  spectra  of  two  different  substances  it  is  desirable  to  be  able  to 
examine  their  spectra  side  by  side.  This  is  provided  for  in  the  better 
forms  of  micro-spectroscopes  by  a  prism  just  below  the  slit,  so  placed 
that  the  light  entering  it  from  a  mirror  at  the  side  of  the  drum  shall  be 
totally  reflected  in  a  vertical  direction,  and  thus  parallel  with  the  rays 
from  the  microscope.  The  two  spectra  will  be  side  by  side  with  a 
narrow  dark  line  separating  them.  If  now  the  slit  is  well  focused  and 
daylight  be  sent  through  the  microscope  and  into  the  side  to  the  reflect- 
ing or  comparison  prism,  the  colored  bands  and  the  Fraunhofer  dark 
lines  will  appear  directly  continuous  across  the  two  spectra.  The 
prism  for  the  comparison  spectrum  is  movable  and  may  be  thrown  en- 
tirely out  of  the  field  if  desired.  When  it  is  to  be  used,  it  is  moved 
about  half  way  across  the  field  so  that  the  two  spectra  shall  have 
about  the  same  width. 

§  202.  Scale  of  "Wave  Lengths. — In  the  Abbe  micro-spectro- 
scope the  scale  is  in  a  separate  tube  near  the  top  of  the  prism  and  at 
right  angles  to  the  prism-tube.  A  special  mirror  serves  to  light  the 
scale,  which  is  projected  upon  the  spectrum  by  a  lens  in  the  scale-tube. 
This  scale  is  of  the  Angstrom  form,  and  the  wave  lengths  of  any  part 
of  the  spectrum  may  be  read  off  directly,  after  the  scale  is  once  set  in 
the  proper  position,  that  is,  when  it  is  set  so  that  any  given  wave 
length  on  the  scale  is  opposite  the  part  of  the  spectrum  known  by  pre- 
vious investigation  to  have  that  particular  wave  length.  The  point 
most  often  selected  for  setting  the  scale  is  opposite  the  sodium  line 
where  the  wave  length  is,  according  to  Angstrom,  0.5892  fji.  In  ad- 


CH.  FT]        MICRO-SPECTROSCOPE  AND  POLARISCOPE  143 

justing  the  scale,  one  may  focus  very  sharply  the  dark  sodium  line  of 
the  solar  spectrum  and  set  the  scale  so  that  the  number  0.589  is  oppo- 
site the  sodium  or  D  line,  or  a  method  that  is  frequently  used  and 
serves  to  illustrate  §  191—2,  is  to  sprinkle  some  salt  of  sodium  (carbon- 
ate of  sodium  is  good)  in  an  alcohol  lamp  flame  and  to  examine  this 
flame.  If  this  is  done  in  a  darkened  place  with  a  spectroscope,  a 
narrow  bright  band  will  be  seen  in  the  yellow  part  of  the  spectrum. 
If  now  ordinary  daylight  is  sent  through  the  comparison  prism,  the 
bright  line  of  the  sodium  will  be  seen  to  be  directly  continuous  with 
the  dark  line  at  D  in  the  solar  spectrum  (Fig.  121).  By  reflecting 
light  into  the  scale-tube  the  image  of  the  scale  will  appear  on  the 
spectrum,  and  by  a  screw  just  under  the  scale- tube  but  within  the 
prism-tube,  the  proper  point  on  the  scale  (0.589  /*)  can  be  brought 
opposite  the  sodium  band.  All  the  scale  will  then  give  the  wave 
lengths  directly.  Sometimes  the  scale  is  oblique  to  the  spectrum. 
This  may  be  remedied  by  turning  the  prism-tube  slightly  one  way  or 
the  other.  It  may  be  due  to  the  wrong  position  of  the  scale  itself.  If 
so,  grasp  the  milled  ring  at  the  distal  end  of  the  scale-tube  and,  while 
looking  into  the  spectroscope,  rotate  the  tube  until  the  lines  of  the  scale 
are  parallel  with  the  Fraunhofer  lines.  It  is  necessarj*  in  adjusting 
the  scale  to  be  sure  that  the  larger  number,  o.  70,  is  at  the  red  end  of 
the  spectrum. 

The  numbers  on  the  scale  should  be  very  clearly  defined.  If  the}* 
do  not  so  appear,  the  scale-tube  must  be  focused  by  grasping  the  outer 
tube  of  the  scale-tube  and  moving  it  toward  or  from  the  prism-tube 
until  the  scale  is  distinct.  In  focusing  the  scale,  grasp  the  outer  scale- 
tube  with  one  hand  and  the  prism-tube  with  the  other,  and  push  or 
pull  in  opposite  directions.  In  this  way  one  will  be  less  liable  to  injure 
the  spectroscope. 

§  203.  Designation  of  Wave  Length. — Wave  lengths  of  light 
are  designated  by  the  Greek  letter  A.,  followed  by  the  number  indicat- 
ing the  wave  length  in  some  fraction  of  a  meter.  With  the  Abbe 
micro- spectroscope  the  micron  is  taken  as  the  unit  as  with  other  micro- 
scopical measurements  (§  166).  Various  units  are  in  use,  as  the  one 
hundred  thousandth  of  a  millimeter,  millionths  or  ten  millionths  of  a 
millimeter.  If  these  smaller  units  are  taken,  the  wave  lengths  will  be 
indicated  either  as  a  decimal  fraction  of  a  millimeter  or  as  whole  num- 
bers. Thus,  according  to  Angstrom,  the  wave  length  of  sodium  light 
is  5892  ten  millionths  mm.,  or  589.2  millionths,  or  58.92  one  hundred 
thousandths,  or  0.5892  one  thousandth  mm.,  or  0.5892  J/u.  The  last 
would  be  indicated  thus,  A.  D  '=  0.5892  yu. 


144  MICRO-SPECTROSCOPE  AND  POLARISCOPE        \CH.  VI 

§  204.  Lighting  for  Micro-specftroscope. — For  opaque  objects 
a  strong  light  should  be  thrown  on  them  either  with  a  concave  mirror 
or  a  condensing  lens.  For  transparent  objects  the  amount  of  the  sub- 
stance and  the  depth  of  color  must  be  considered.  As  a  general  rule 
it  is  well  to  use  plenty  of  light,  as  that  from  an  Abbe  illuminator  with 
a  large  opening  in  the  diaphragm,  or  with  the  diaphragm  entirely 
open.  For  very  small  objects  and  thin  layers  of  liquids  it  may  be 
better  to  use  less  light.  One  must  try  both  methods  in  a  given  case, 
and  learn  by  experience. 

The  direct  and  the  comparison  spectra  should  be  about  equally 
illuminated.  One  can  manage  this  by  putting  the  object  requiring  the 
greater  amount  of  illumination  on  the  stage  of  the  microscope  and 
lighting  it  with  the  Abbe  illuminator.  In  lighting  it  is  found  in  gen- 
eral that  for  red  or  yellow  objects,  lamp- light  gives  very  satisfactory 
results.  For  the  examination  of  blood  and  blood  crystals  the  light 
from  a  petroleum  lamp  is  excellent.  For  objects  with  much  blue  or 
violet,  daylight  or  artificial  light  rich  in  blue  light  is  best. 

Furthermore,  one  should  be  on  his  guard  against  confusing  the 
ordinary  absorption  bands  with  the  Fraunhofer  lines  when  daylight  is 
used.  With  lamp-light  the  Fraunhofer  lines  are  absent  and,  therefore, 
not  a  source  of  possible  confusion. 

§  205.  Objectives  to  Use  with  the  Micro-spectroscope. — If 
the  material  is  of  considerable  bulk,  a  low  objective  (16  to  50  mm.)  is 
to  be  preferred.  This  depends  on  the  nature. of  the  object  under  ex- 
amination, however.  In  case  of  individual  crystals  one  should  use 
sufficient  magnification  to  make  the  real  image  of  the  crystal  entirely 
fill  the  width  of  the  slit.  The  length  of  the  slit  may  then  be  regulated 
by  the  screw  on  the  side  of  the  drum,  and  also  by  the  comparison 
prism.  If  the  object  does  not  fill  the  whole  slit  the  white  light  enter- 
ing the  spectroscope  with  the  light  from  the  object  might  obscure  the 
absorption  bands.  For  opaque  objects  illuminating  objectives  are 
useful  (§28,  233.) 

In  using  high  objectives  with  the  micro-spectroscope  one  must 
very  carefully  regulate  the  light  (  Ch.  II)  and  sometimes  shade  the 
object. 

§  206.  Focusing  the  Objective. — For  focusing  the  objective  the 
prism-tube  is  swung  aside,  and  then  the  slit  made  wide  by  turning  the 
adjusting  screw  at  the  side.  If  the  slit  is  open  one  can  see  objects 
when  the  microscope  is  focused  as  with  an  ordinary  ocular.  After  an 
object  is  focused,  it  may  be  put  exactly  in  position  to  fill  the  slit  of  the 


CH.  VF]        MICRO-SPECTROSCOPE  AND  POLARISCOPE  145 

spectroscope,  then  the  knife  edges  are  brought  together  till  the  slit  is 
of  the  right  width  ;  if  the  slit  is  then  too  long  it  may  be  shortened  by 
using  one  of  the  mechanism  screws  on  the  side,  or  if  that  is  not  suffi- 
cient, by  bringing  the  comparison  prism  farther  over  the  field.  If  one 
now  replaces  the  Amici  prism  and  looks  into  the  microscope,  the 
spectrum  is  liable  to  have  longitudinal  shimmering  lines.  To  get  rid 
of  these  focus  up  or  down  a  little  so  that  the  microscope  will  be 
slightly  out  of  focus. 

§  207.  Amount  of  Material  Necessary  for  Absorption  Spectra 
and  its  Proper  Manipulation. — The  amount  of  material  necessary  to 
give  an  absorption  spectrum  varies  greatly  with  different  substances, 
and  can  be  determined  only  by  trial.  It  a  transparent  solid  is  under 
investigation  it  is  well  to  have  it  in  the  form  a  wedge,  then  succes- 
sive thicknesses  can  be  brought  under  the  microscope.  If  a  liquid  sub- 
stance is  being  examined,  a  watch  glass  with  sloping  sides  forms  an 
excellent  vessel  to  contain  it,  then  successive  thicknesses  of  the  liquid 
can  be  brought  into  the  field  as  with  the  wedge-shaped  solid.  Fre- 
quently only  a  very  weak  solution  is  obtainable  ;  in  this  case  it  can  be 
placed  in  a  homoeopathic  vial,  or  in  some  glass  tubing  sealed  at  the 
end,  then  one  can  look  lengthwise  through  the  liquid  and  get  the 
effect  of  a  more  concentrated  solution.  For  minute  bodies  like  crystals 
or  blood  corpuscles,  one  may  proceed  as  described  in  the  previous 
section. 

MICRO-SPECTROSCOPE — EXPERIMENTS* 

§  208.  Put  the  micro-spectroscope  in  position,  arrange  the  slit 
and  the  Amici  prism  so  that  the  spectrum  will  show  the  various  spec- 
tral colors  going  directly  across  it  (§  198-199)  and  carefully  focus  the 
slit.  This  may  be  done  either  by  swinging  the  prism-tube  aside  and 
proceeding  as  for  the  ocular  micrometer  (§  172),  or  by  moving  the 
eye- lens  of  the  ocular  up  and  down  while  looking  into  the  micro- 
spectroscope  until  the  dark  lines  of  the  solar  spectrum  are  distinct.  If 
they  cannot  be  made  distinct  by  focusing  the  slit,  then  the  light  is  too 
feeble  or  the  slit  is  too  wide  (§  198).  With  the  lever  move  the  com- 
parison prism  across  half  the  field  so  that  the  two  spectra  shall  be  of 
about  equal  width.  For  lighting,  see  §  204. 


*If  one  does  not  possess  a  micro-spectroscope,  quite  satisfactory  results  may  be 
obtained  by  using  a  microscope  with  a  16  to  12  mm.  objective  and  a  pocket,  direct- 
vision  spectroscope  in  place  of  the  eye-piece.  (Bleile,  Trans.  Amer.  Micr.  Soc. 
1900,  p.  8). 


146  MICRO-SPECTROSCOPE  AND  POLARISCOPE        {CH.  VI 

§  209.  Absorption  Spectrum  of  Permanganate  of  Potash.— 
Make  a  solution  of  permanganate  of  potash  in  water  of  such  a  strength 
that  a  stratum  3  or  4  mm.  thick  is  transparent.  Put  this  solution  in  a 
watch-glass  with  sloping  sides,  and  put  it  under  the  microscope.  Use 
a  50  mm.  or  16  mm.  objective,  and  use  the  full  opening  of  the  illumi- 
nator. Light  strongly.  Look  into  the  spectroscope  and  slowly  move 
the  watch-glass  into  the  field.  Note  carefully  the  appearance  with  the 
thin  stratum  of  liquid  at  the  edge  and  then  as  it  gradually  thickens  on 
moving  the  watch-glass  still  farther  along.  Count  the  absorption 
bands  and  note  particularly  the  red  and  blue  ends.  Compare  carefully 
with  the  comparison  spectrum  (Figs.  121,  122).  For  strength  of  solu- 
tion see  §  207. 

§  210.  Absorption  Spectrum  of  Blood. — Obtain  blood  from  a 
recently  killed  animal,  or  flame  a  needle,  and  after  it  is  cool  prick  the 
finger  two  or  three  times  in  a  small  area,  then  wind  a  handkerchief  or 
a  rubber  tube  around  the  base  of  the  finger,  and  squeeze  the  finger 
with  the  other  hand.  Some  blood  will  ooze  out  of  the  pricks.  Rinse  this 
off  into  a  watch-glass  partly  filled  with  water.  Continue  to  add  the  blood 
until  the  water  is  quite  red.  Place  the  watch-glass  of  diluted  blood  un- 
der the  microscope  in  place  of  the  permanganate,  using  the  same  object- 
ive, etc.  Note  carefully  the  spectrum.  It  would  be  advantageous  to 
determine  the  wave  length  opposite  the  center  of  the  dark  bands.  This 
may  easily  be  done  by  setting  the  scale  properly  as  described  in  §  202. 
Make  another  preparation,  but  use  a  homeopathic  vial  instead  of  a 
watch-glass.  Cork  the  vial  and  lay  it  down  upon  the  stage  of  the 
microscope.  Observe  the  spectrum.  It  will  be  like  that  in  the  watch- 
glass.  Remove  the  cork  and  look  through  the  whole  length  of  the  vial. 
The  bands  will  be  much  darker,  and  if  the  solution  is  thick  enough 
only  red  and  a  little  orange  will  appear.  Re-insert  the  cork  and  incline 
the  vial  so  that  the  light  traverses  a  very  thin  layer,  then  gradually 
elevate  the  vial  and  the  effect  of  a  thicker  and  thicker  layer  may  be 
seen.  Note  especially  that  the  two  characteristic  bands  unite  and 
form  one  wide  band  as  the  stratum  of  liquid  thickens.  Compare  with 
the  following  : 

Add  to  the  vial  of  diluted  blood  a  drop  or  two  of  ammonium  sul- 
phide, such  as  is  used  for  a  reducing  agent  in  chemical  laboratories. 
Shake  the  bottle  gently  and  then  allow  it  to  stand  for  ten  or  fifteen 
minutes.  Examine  it  and  the  two  bands  will  have  been  replaced  by  a 
single,  less  clearly  defined  band  in  .about  the  same  position.  The 
blood  will  also  appear  somewhat  purple.  Shake  the  vial  vigorously 


CH.  VI}        MICRO-SPECTROSCOPE  AND  POLARISCOPE  147 

and  the  color  will  change  to  the  bright  red  of  fresh  blood.  Examine 
it  again  with  the  spectroscope  and  the  two  bands  will  be  visible.  After 
five  or  ten  minutes  another  examination  will  show  but  a  single  band. 
Incline  the  bottle  so  that  a  thin  stratum  may  be  examined.  Note 
that  the  stratum  of  liquid  must  be  considerably  thicker  to  show  the 
absorption  band  than  was  necessary  to  show  the  two  bands  in  the  first 
experiment.  Furthermore,  while  the  single  band  may  be  made  quite 
black  on  thickening  the  stratum,  it  will  not  separate  into  two  bands 
with  a  thinner  stratum.  In  this  experiment  it  is  very  instructive  to 
have  a  second  vial  of  fresh  diluted  blood,  say  that  from  the  watch- 
glass,  before  the  opening  of  the  comparison  prism.  The  two  banded 
spectrum  will  then  be  in  position  to  be  compared  with  the  spectrum  of 
the  blood  treated  with  the  ammonium  sulphide. 

The  two  banded  spectrum  is  that  of  oxy-hemoglobin,  or  arterial  blood, 
the  single  banded  spectrum  of  hemoglobin  (sometimes  called  reduced 
hemoglobin)  or  venous  blood,  that  is,  the  respiratory  oxygen  is  present 
in  the  two  banded  spectrum  but  absent  from  the  single  banded  spectrum. 
When  the  bottle  was  shaken  the  hemoglobin  took  up  oxygen  from  the 
air  and  became  oxy-hemoglobin,  as  occurs  in  the  lungs,  but  soon  the 
ammonium  sulphide  took  away  the  respiratory  oxygen,  thus  reducing 
the  oxy-hemoglobin  to  hemoglobin.  This  may  be  repeated  many 
times  (Fig.  122). 

§  211.  Met-Hemoglobin. — The  absorption  spectrum  of  met- 
hemoglobin  is  characterized  by  a  considerable  darkening  of  the  blue 
end  of  the  spectrum  and  of  four  absorption  bands,  one  in  the  red  near 
the  line  C  and  two  between  D  and  E,  nearly  in  the  place  of  the  two 
bands  of  oxy-hemoglobin  ;  finally  there  is  a  somewhat  faint,  wide 
band  near  F.  Such  a  met-hemoglobin  spectrum  is  best  obtained  by 
making  a  solution  of  blood  in  water  of  such  a  concentration  that  the 
two  oxy-hemoglobin  bands  run  together  (§  210),  'and  then  adding 
three  or  four  drops  of  a  y1^  per  cent,  aqueous  solution  of  permanganate 
of  potash.  Soon  the  bright  red  will  change  to  a  brownish  color,  when 
it  may  be  examined  (Fig.  121).  Instead  of  the  permanganate  one 
may  use  hydrogen  dioxide  (H2O2). 

§  212.  Carbon  Monoxide  Hemoglobin  (CO-Hemoglobin). — 
To  obtain  this,  kill  an  animal  in  illuminating  gas,  or  one  may  allow 
illuminating  gas  to  bubble  through  some  blood  already  taken  from  the 
body.  The  gas  should  bubble  through  a  minute  or  two.  The  oxygen 
will  be  displaced  by  carbon  monoxide.  This  forms  quite  a  stable  com- 
pound with  hemoglobin,  and  is  of  a  bright  cherry-red  color.  Its 


148  MICRO-SPECTROSCOPE  AND  POLARISCOPE        [CH.  VI 

spectrum  is  nearly  like  that  of  oxy-hemoglobin,  but  the  bands  are 
farther  toward  the  blue.  Add  several  drops  of  ammonium  sulphide  and 
allow  the  blood  to  stand  some  time.  No  reduction  will  take  place, 
thus  forming  a  marked  contrast  to  solutions  of  oxy-hemoglobin.  By 
the  addition  of  a  few  drops  of  glacial  acetic  acid  a  dark  brownish  red 
color  is  produced. 

§  213.  Carmine  Solution. — Make  a  solution  of  carmine  by  put- 
ting y^th  gram  of  carmine  in  100  cc.  of  water  and  adding  10  drops  of 
strong  ammonia.  Put  some  of  this  in  a  watch-glass  or  in  a  small  vial 
and  compare  the  spectrum  with  that  of  oxy-hemoglobin  or  carbon 
monoxide  hemoglobin.  It  has  two  bands  in  nearly  the  same  position, 
thus  giving  the  spectrum  a  striking  similarity  to  blood.  If  now  several 
drops,  15  or  20,  of  glacial  acetic  acid  are  added  to  the  carmine,  the 
bands  remain  and  the  color  is  not  markedly  changed,  while  with 
either  oxy-hemoglobin  or  CO-hemoglobin  the  color  would  be  de- 
cidedly changed  from  the  bright  red  to  a  dull  reddish  brown,  and 
the  spectrum,  if  any  could  be  seen,  would  be  markedly  different. 
Carmine  and  O- hemoglobin  can  be  distinguished  by  the  use  of  ammo- 
nium sulphide,  the  carmine  remaining  practically  unchanged  while  the 
blood  shows  the  single  band  of  hemoglobin  (§  210).  The  acetic  acid 
serves  to  differentiate  the  CO-hemoglobin  as  well  as  the  O-hemoglobin. 

§  214.  Colored  Bodies  not  giving  Distinctly  Banded  Absorp- 
tion Specftra. — Some  quite  brilliantly  colored  objects,  like  the  skin  of 
a  red  apple,  do  not  give  a  banded  spectrum.  Take  the  skin  of  a  red 
apple,  mount  it  on  a  slide,  put  on  a  cover-glass  and  add  a  drop  of 
water  at  the  edge  of  the  cover.  Put  the  preparation  under  the  micro- 
scope and  observe  the  spectrum.  Although  no  bands  will  appear,  in 
some  cases  at  least,  yet  the  ends  of  the  spectrum  will  be  restricted  and 
various  regions  of  the  spectrum  will  not  be  so  bright  as  the  comparison 
spectrum.  Here  the  red  color  arises  from  the  mixture  of  the  unab- 
sorbed  waves,  as  occurs  with  other  colored  objects.  In  this  case, 
however,  not  all  the  light  of  a  given  wave  length  is  absorbed,  conse- 
quently there  are  no  clearly  defined  dark  bands,  the  light  is  simply 
less  brilliant  in  certain  regions  and  the  red  rays  so  predominate  that 
they  give  the  prevailing  color. 

§  215.  Nearly  Colorless  Bodies  with  Clearly  Marked  Ab- 
sorption Spectra. — In  contradistinction  to  the  brightly  colored 
objects  with  no  distinct  absorption  bands  are  those  nearly  colorless 
bodies  and  solutions  which  give  as  sharply  defined  absorption  bands  as 
could  be  desired.  The  best  examples  of  this  are  afforded  by  solutions 


CH.   VI]        MICRO-SPECTROSCOPE  AND  POLARISCOPE  149 

of  the  rare  earths,  didymium,  etc.  These  in  solutions  that  give 
hardly  a  trace  of  color  to  the  eye  give  absorption  bands  that  almost 
rival  the  Fraunhofer  lines  in  sharpness. 

§  216.  Absorption  Spectra  of  Minerals. — As  example  take  some 
monazite  sand  on  a  slide  and  either  mount  it  in  balsam  (see  §  256), 
or  cover  and  add  a  drop  of  water.  The  examination  may  be  made  also 
with  the  dry  sand,  but  it  is  less  satisfactory.  Light  well  with  trans- 
mitted light,  and  move  the  preparation  slowly  around.  Absorption 
bands  will  appear  occasionally.  Swing  the  prism  tube  off  the  ocular, 
open  the  slit  and  focus  the  sand.  Get  the  image  of  one  or  more  grains 
directly  in  the  slit,  then  narrow  and  shorten  the  slit  so  that  no  light 
can  reach  the  spectroscope  that  has  not  traversed  the  grain  of  sand. 
The  spectrum  will  be  satisfactory  under  such  conditions.  It  is 
frequently  of  great  service  in  determining  the  character  of  unknown 
mineral  sands  to  compare  the  spectra  with  known  minerals.  If  the 
absorption  bands  are  identical,  it  is  strong  evidence  in  favor  of  the 
identity  of  the  minerals.  For  proper  lighting  see  §  204. 

§  217.  While  the  study  of  absorption  spectra  gives  one  a  great 
deal  of  accurate  information,  great  caution  must  be  exercised  in  draw- 
ing conclusions  as  to  the  identity  or  even  the  close  relationship  of 
bodies  giving  approximately  the  same  absorption  spectra.  The  rule 
followed  by  the  best  workers  is  to  have  a  known  body  as  control  and 
to  treat  the  unknown  body  and  known  body  with  the  same  reagents, 
and  to  dissolve  them  in  the  same  medium.  If  all  the  reactions  are 
identical  then  the  presumption  is  strong  that  the  bodies  are  ident- 
ical or  very  closely  related.  For  example,  while  one  might  be  in  doubt 
between  a  solution  of  oxy-  or  CO-hemoglobin  and  carmine,  the  addition 
of  ammonium  sulphide  serves  to  change  the  double  to  a  single  band 
in  the  O-hemoglobin,  and  glacial  acetic  acid  enables  one  to  distinguish 
between  the  CO-blood  and  the  carmine,  although  the  ammonium  sul- 
phide would  not  enable  one  to  make  the  distinction.  Furthermore  it 
is  unsafe  to  compare  objects  dissolved  in  different  media.  The  same 
objects  as  "cyanine  and  aniline  blue  dissolved  in  alcohol  give  a  very 
similar  spectrum,  but  in  water  a  totally  different  one."  "Totally  dif- 
ferent bodies  show  absorption  bands  in  exactly  the  same  position  (solid 
nitrate  of  uranium  and  permanganate  of  potash  in  the  blue)."  (Mac- 
Munn).  The  rule  given  by  MacMunn  is  a  good  one  :  "The  recogni- 
tion of  a  body  becomes  more  certain  if  its  spectrum  consists  of  several 
absorption  bands,  but  even  the  coincidence  of  these  bands  with  those 
of  another  body  is  not  sufficient  to  enable  us  to  infer  chemical  identity; 


150  MICRO-SPECTROSCOPE  AND  POLARISCOPE        [CH.  VI 

what  enables  us  to  do  so  with  certainty  is  the  fact  :  that  the  two  solu- 
tions give  bands  of  equal  intensities  in  the  same  parts  of  the  spectrum 
which  undergo  analogous  changes  on  the  addition  of  the  same  reagent. ' ' 

REFERENCES   TO   THE   MICRO-SPECTROSCOPE   AND 
SPECTRUM     ANALYSIS 

The  micro-spectroscope  is  playing  an  ever-increasingly  important  role  in  the 
spectrum  analysis  of  animal  and  vegetable  pigments,  and  of  colored  mineral  and 
chemical  substances,  therefore  a  somewhat  extended  reference  to  literature  will  be 
given.  Full  titles  of  the  books  and  periodicals  will  be  found  in  the  Bibliography 
at  the  end. 

Angstrom,  Recherches  sur  le  spectre  solaire,  etc.  Also  various  papers  in 
periodicals.  See  Royal  Soc's  Cat'l  Scientific  Papers  ;  Anthony  &  Brackett  ;  Beale, 
p.  269  ;  Behrens,  p.  139  ;  Kossel  und  Schiefferdecker,  p.  63  ;  Carpenter,  p.  323  ; 
Browning,  How  to  Work  with  the  Spectroscope,  and  in  Monthly  Micr.  Jour.,  II, 
p.  65  ;  Daniell,  Principles  of  Physics.  The  general  principles  of  spectrum  analysis 
are  especially  well  stated  in  this  work,  pp.  435-455  ;  Davis,  p.  342  ;  Dippel,  p. 
277  ;  Frey  ;  Gamgee,  p.  91  ;  Halliburton  ;  Hogg,  p.  122  ;  also  in  Monthly  Micr. 
Jour.,  Vol.  II,  on  colors  of  flowers;  Jour,  Roy.  Micr.  Soc.,  1880,  1883,  and  in 
various  other  vols. ;  Kraus  ;  Lockyer  ;  M'Kendrick  ;  MacMunn  ;  and  also  in 
Philos,  Trans.  R.  S.,  1886;  various  vols.  of  Jour.  Physiol.;  Nageli  und  Schwend- 
ener  ;  Proctor;  Ref.  Hand-Book  Med.  Science,  Vol.  I,  p.  577,  VI,  p.  516,  VII,  p. 
426;  Roscoe  ;  Schellen  ;  Sorby,  in  Beale,  p.  269;  also  Proc.  R.  S.,  1874,  p.  31, 
1867,  p.  433  ;  see  also  in  the  Scientific  Review,  Vol.  V,  p.  66,  Vol.  II,  p.  419. 
The  larger  works  on  Physiology,  Chemistry  and  Physics  may  also  be  consulted 
with  profit. 

Vogel,  Spectrum  analysis  ;  also  in  Nature,  Vol.  xix,  p.  495,  on  absorption  spec- 
tra. The  bibliography  in  MacMunn  is  excellent  and  extended. 

For  hemochromogen  in  medico-legal  cases  see  Bleile,  Trans.  Amer.  Micr. 
Soc.,  1900,  p.  9. 

MICRO-POLARISCOPB 

g  218.  The  micro-polariscope,  or  polarizer,  is  a  polariscope  used  in  connection 
with  a  microscope. 

The  most  common  and  typical  form  consists  of  two  Nicol  prisms,  that  is,  two 
somewhat  elongated  rhombs  of  Iceland  spar  cut  diagonally  and  cemented  together 
with  Canada  balsam.  These  Nicol  prisms  are  then  mounted  in  such  a  way  that 
the  light  passes  through  them  lengthwise,  and  in  passing  is  divided  into  two  rays 
of  plane  polarized  light.  The  one  of  these  rays  obeying  most  nearly  the  ordinary 
law  of  refraction  is  called  the  ordinary  ray,  the  one  departing  farthest  from  the 
law  is  called  the  extra-ordinary  ray.  These  two  rays  are  not  only  polarized,  but 
polarized  in  planes  almost  exactly  at  right  angles  to  each  other.  The  Nicol  prism 
totally  reflects  the  ordinary  ray  at  the  cemented  surface  as  it  meets  that  surface  at 
an  angle  greater  than  the  critical  angle,  and  only  the  extraordinary  or  less  refracted 
ray  is  transmitted. 


CH.  VI~\        MICRO-SPECTROSCOPE  AND  POLARISCOPE  151 

§  219.  Polarizer  and  Analyzer. — The  polarizer  is  one  of  the  Nicol  prisms.  It  is 
placed  beneath  the  object  and  in  this  way  the  object  is  illuminated  with  polarized 
light.  The  analyzer  is  the  other  Nicol  and  is  placed  at  some  level  above  the  object, 
very  conveniently  above  the  ocular. 

When  the  corresponding  faces  of  the  polarizer  and  analyzer  are  parallel  i.  e., 
when  the  faces  through  which  the  oblique  section  passes  are  parallel,  light  passes 
freely  through  the  analyzer  to  the  eye.  If  these  corresponding  faces  are  at  right 
angles,  that  is,  if  the  Nicols  are  crossed,  then  the  light  is  entirely  cut  off  and  the 
two  transparent  prisms  become  opaque  to  ordinary  light.  There  are  then,  in  the 
complete  revolution  of  the  analyzer,  two  points,  at  o°  and  180°,  where  the  corre- 
sponding faces  are  parallel  and  where  light  freely  traverses  the  analyzer.  There 
are  also  two  crossing  points  of  the  Nicols,  at  90°  and  270°,  where  the  light  is  extin- 
guished. In  the  intermediate  points  there  is  a  sort  of  twilight. 

\  220.  Putting  the  Polarizer  and  Analyzer  in  Position. — Swing  the  diaphragm 
carrier  of  the  Abbe  illuminator  out  from  under  the  illuminator,  remove  the  disk 
diaphragm  or  open  widely  the  iris  diaphragm  and  place  the  analyzer  in  the  dia- 
phragm carrier,  then  swing  it  back  under  the  illuminator.  Remove  the  ocular, 
put  the  graduated  ring  on  the  top  of  the  tube  and  then  replace  the  ocular  and  put 
the  analyzer  over  the  ocular  and  ring.  Arrange  the  graduated  ring  so  that  the  indi- 
cator shall  stand  at  o°  when  the  field  is  lightest.  This  may  be  done  by  turning  the 
tube  down  so  that  the  objective  is  near  the  illuminator,  then  shading  the  stage  so 
that  none  but  polarized  light  shall  enter  the  microscope.  Rotate  the  analyzer  until 
the  lightest  possible  point  is  found,  then  rotate  the  graduated  ring  till  the  index 
stands  at  o°.  The  ring  may  then  be  clamped  to  the  tube  by  the  side  screw  for  the 
purpose.  Or,  more  easily,  one  may  set  the  index  at  o°,  clamp  the  ring  to  the 
microscope,  then  rotate  the  draw-tube  of  the  microscope  till  the  field  is  lightest. 

I  221.  Adjustment  of  the  Analyzer. — The  analyzer  should  be  capable  of 
moving  up  and  down  in  its  mounting,  so  that  it  can  be  adjusted  to  the  eye-point 
of  the  ocular  with  which  it  is  used.  If  on  looking  into  the  analyzer  with  parallel 
Nicols  the  edge  of  the  field  is  not  sharp,  or  if  it  is  colored,  the  analyzer  is  not  in 
a  proper  position  with  reference  to  the  eye-point,  and  should  be  raised  or  lowered 
till  the  edge  of  the  field  is  perfectly  sharp  and  as  free  from  color  as  the  ocular 
with  the  analyzer  removed. 

§  222.  Objectives  to  Use  with  the  Polariscope. — Objectives  of  the  lowest  pow- 
ers may  be  used,  and  also  all  intermediate  forms  up  to  a  2  mm.  homogeneous  im- 
mersion. Still  higher  objectives  may  be  used  if  desired.  In  general,  however, 
the  lower  powers  are  somewhat  more  satisfactory.  A  good  rule  to  follow  in  this 
case  is  the  general  rule  in  all  microscopic  work, — use  the  power  that  most  clearly 
and  satisfactorily  shows  the  object  under  investigation. 

$  223.  Lighting  for  Micro- Polariscope  Work. — Follow  the  general  directions 
given  in  Chapter  II.  It  is  especially  necessary  to  shade  the  object  so  that  no  un- 
polarized  light  can  enter  the  objective,  otherwise  the  field  cannot  be  sufficiently 
darkened.  No  diaphragm  is  used  over  the  polarizer  for  most  examinations.  Direct 
sunlight  may  be  used  to  advantage  with  some  objects,  and  as  a  rule  the  object 
would  best  be  very  transparent. 

\  224.  Mounting  Objects  for  the  Polariscope. — So  far  as  possible  objects 
should  be  mounted  in  balsam  to  render  them  transparent.  In  many  cases  objects 
mounted  in  water  do  not  give  satisfactory  polariscope  appearances.  For  example, 


152  MICRO-SPECTROSCOPE  AND  POLARISCOPE        \_CH.  VI 

if  starch  is  mounted  dry  or  in  water,  the  appearances  are  not  so  striking  as  in  a 
balsam  mount  (Davis,  p.  337  ;  Suffolk). 

\  225.  Purpose  of  a  Micro-Polariscope. — The  object  of  a  micro-polariscope  is 
to  determine,  in  microscopic  masses,  one  or  more  of  the  following  points  :  (A) 
Whether  the  body  is  singly  refractive,  mono-refringent,  or  isotropic,  that  is,  opti- 
cally homogeneous,  as  are  glass  and  crystals  belonging  to  the  cubical  system  ;  (B) 
Whether  the  object  is  doubly  refractive  or  anisotropic,  uniaxial  or  biaxial  ;  (C) 
Pleochroism  ;  (D)  The  rotation  of  the  plane  of  polarization,  as  with  solutions  of 
sugar,  etc.  ;  (E)  To  aid  in  petrology  and  mineralogy  ;  (F)  To  aid  in  the  determi- 
nation of  very  minute  quantities  of  crystallizable  substances;  (G)  For  the  pro- 
duction of  colors. 

For  petrological  and  mineralogical  investigations  the  microscope  should  possess 
a  graduated,  rotating  stage  so  that  the  object  can  be  rotated,  and  the  exact  angle 
of  rotation  determined.  Fig.  126.  It  is  also  found  of  advantage  in  investigating 
object  swith  polarized  light  where  colors  appear,  to  combine  a  polariscopic  and 
spectroscope  ( Spectro-Polariscope ) . 

MICRO-POLARISCOPE — EXPERIMENTS 

§  226.    Arrange  the  polarizer  and  analyzer  as  directed  above  (§  220) 
and  use  a  16  mm.  objective  except  when  otherwise  directed. 

(A)  Isotropic  or  Singly  Refracting  Objects. — Light  the  mi- 
croscope well  and  cross  the  Nicols,  shade  the  stage  and  make  the  field 
as  dark  as  possible  (§219).     As  an  isotropic  substance,  put  an  ordin- 
ary glass  slide  under  the  microscope.     The  field  will  remain  dark.     As 
an  example  of  a  crystal  belonging  to  the  cubical  system  and  hence  iso- 
tropic, make    a   strong  solution  of  common  salt    (sodium    chloride) 
put   a   drop   on   a   slide  and    allow  it  to  crystallize,  put  it  under  the 
microscope,  remove  the  analyzer,  focus  the  crystals  and  then  replace 
the  analyzer  and  cross  the  Nicols.     The  field  and  the  crystals  will  re- 
main dark. 

(B)  Anisotropic  or  Doubly  Refracting  Objects. — Make  a  fresh 
preparation  of  carbonate  of  lime  crystals  like  that  described  for  pedesis 
(§  !50»  or  use  a  preparation  in  which  the  crystals  have  dried  to  the 
slide,  use  a  5  or  3  mm.   objective,  shade  the  object  well,  remove  the 
analyzer  and  focus  the  crystals,  then  replace  the  analyzer.     Cross  the 
Nicols.     In  the  dark  field  will  be  seen  multitudes  of  shining  crystals, 
and  if  the  preparation  is  a  fresh  one  in  water,   part   of  the  smaller 
crystals  will  alternately  flash  and  disappear.      By  observing  carefully, 
some  of  the  larger  crystals  will  be  found  to  remain  dark  with  crossed 
Nicols,  others  will  shine  continuously.     If  the  crystals  are  in  such   a 
position  that  the  light  passes  through  them  parallel  with  the  optic 


FIG.  126.  Chamofs  Microscope 
for  Micro-Chemical  Analysis  (Jour- 
nal of  Applied  Microscopy,  1809,  p. 


This  is  a  modified  and  simplified 
petrographical  microscope  and  has 
all  the  attachments  and  motions  nec- 
essary for  micro-chemical  analysis, 
As  the  objects  studied  are  mostly 
liquid  or  in  liquids  the  microscope 
has  no  joint  as  it  must  be  used  in  a 
vertical  position. 


154  MICRO-SPECTROSCOPE  AND  POLARISCOPE        [CH.  VI 

axis,*  the  crystals  are  isotropic  like  salt  crystals  and  remain  dark. 
If,  however,  the  light  traverses  them  in  any  other  direction  the  ray 
from  the  polarizer  is  divided  into  two  constituents  vibrating  in  planes 
at  right  angles  to  each  other,  and  one  of  these  will  traverse  the  an- 
alyzer, hence  such  crystals  will  appear  as  if  self-luminous  in  a  dark- 
field.  The  experiment  with  these  crystals  from  the  frog  succeeds  well 
with  a  2  mm.  homogeneous  immersion. 

As  a  further  illustration  of  anisotropic  objects,  mount  some  cotton 
fibers  in  balsam  (§  256),  also  some  of  the  lens  paper  (§  114;.  These 
furnish  excellent  examples  of  vegetable  fibers. 

Striated  muscle  fibers  are  also  very  well  adapted  for  polarizing 
objects. 

.  As  examples  of  biaxial  crystals,  allow  some  borax  solution  to  dry 
and  crystallize  on  a  slide  ;  use  the  crystals  as  objects.  As  all  doubly 
refracting  objects  restore  the  light  with  crossed  Nicols,  they  are  some- 
times called  depolarizing. 

(C)  Pleochroism. — This  is  the  exhibition  of  different  tints  as  the 
analyzer  is  rotated.  An  excellent  subject  for  this  will  be  found  in 
blood  crystals. 

§  227.  Production  of  Colors. — For  the  production  of  gorgeous 
colors,  a  plate  of  selenite  giving  blue  and  yellow  colors  is  placed  between 
the  polarizer  and  the  object.  If  properly  mounted,  the  selenite  is  very 
conveniently  placed  on  the  diaphragm  carrier  of  the  Abbe  illuminator, 
just  above  the  polarizer.  A  thin  plate  or  film  of  mica  also  answers 
well. 

It  is  not  necessary  to  use  selenite  or  mica  for  the  production  of  the 
most  glorious  colors  in  many  objects.  One  of  the  most  beautiful  pre- 
parations, and  one  of  the  most  instructive  also,  may  be  prepared  as 
follows  :  Heat  some  xylene  balsam  on  a  slide  until  the  xylene  is  nearly 
evaporated.  Add  some  crystals  of  the  medicine,  sulphonal  and  warm 
till  the  sulphonal  is  melted  and  mixes  with  the  balsam.  While  the 
balsam  is  still  melted  put  on  a  cover-glass.  If  one  gets  perfect  crystals 
there  will  be  shown  not  only  the  most  beautiful  colors,  but  the  black 
cross  with  perfection.  (Clark). 


*The  optic  axis  of  doubly  refracting  crystals  is  the  axis  along  which  the  crystal 
is  not  doubly  refracting,  but  isotropic  like  glass.  When  there  is  but  one  such 
axis,  the  crystal  is  said  to  be  uniaxial,  if  there  are  two  such  axes  the  crystal  is 
said  to  be  bi-axial. 

The  crystals  of  carbonate  of  lime  from  the  frog  (see  \  151)  are  uniaxial  crystals. 
Borax  crystals  are  bi-axial. 


CH.    VI~\  MICRO-CHEMISTRY  155 

It  is  very  instructive  and  interesting  to  examine  many  organic 
and  inorganic  substances  with  a  micro-polarizer. 

REFERENCES  TO  THE   POLARISCOPE   AND    TO  THE   USE   OF  POLARIZED 

LIGHT. 

Anthony  &  Brackett,  133  ;  Behrens  ;  Behrens,  Kossel  und  Schiefferdecker  ; 
Carnoy,  61  ;  Carpenter-Dallinger,  317,  1097;  Clark;  Daniel,  494;  Davis;  v. 
Ebener  ;  Gamgee  ;  Halliburton,  36,272  ;  Hogg,  133,729  ;  Lehmann  ;  M'Kendrick  ; 
Nageli  undSchwendener,  299  ;  Quekett  ;  Suffolk,  125  ;  Valentin  ;  Physical  Review, 
I.,  p.  127.  Daniell,  Physics  for  Medical  Students.  Nichols,  Physics. 

MICRO-CHEMISTRY 

§  228.  During  the  last  decade  the  microscope  has  become  one  of 
the  necessities  of  the  expert  chemist,  and  the  signs  of  the  times 
indicate  that  in  every  research  laboratory  of  chemistry  the  microscope 
will  become  as  familiar  as  it  now  is  in  research  laboratories  of  biology. 
Its  proper  place  in  chemistry  has  been  admirably  stated  by  Chamot : 

"It  is  rather  remarkable  how  slow  American  chemists  have  been  in  realizing 
the  importance  of  the  microscope  as  an  adjunct  to  every  chemical  laboratory. 
This  is,  perhaps,  largely  due  to  the  fact  that  few  of  our  students  in  chemistry 
become  familiar  with  the  construction  and  manipulation  of  this  instrument,  just 
as  few  of  them  become  sufficiently  familiar  with  the  spectroscope  and  its  manifold 
uses  ;  and  doubtless  also  because  of  the  prevailing  impression  that  a  microscope 
is  primarily  an  instrument  for  the  biologist  and  is  of  necessity  a  most  expensive 
luxury.  The  fact  is,  however,  that  this  instrument  is  now  far  from  being  a  luxury 
to  the  chemist,  and  the  time  is  not  far  distant  when  it  will  be  conceded  to  be  as 
much  a  necessity  in  every  analytical  laboratory  as  is  the  balance. 

"Nor  is  the  apprenticeship  to  its  use  in  chemical  work  long  or  intricate. 

"Micro- chemical  analysis  should  appeal  to  every  chemist  because  of  its  neat- 
ness, wonderful  delicacy,  in  which  it  is  not  excelled  even  by  the  spectroscope,  and 
the  expedition  with  which  an  analysis  can  be  made.  A  complete  analysis, 
intricate  though  it  may  be,  is  a  matter  of  a  few  minutes  rather  than  of  a  few  hours. 

"While  there  is  no  good  reason  to  believe,  as  do  some  enthusiasts,  that  this 
new  system  is  to  displace  the  old  analysis  in  the  wet  way,  every  chemist  should, 
nevertheless,  familiarize  himself  with  the  microscope,  its  accessories,  and  the 
elegant  and  time-saving  methods  of  micro-analysis,  thus  enabling  him  to  examine 
qualitatively  the  most  minute  amounts  of  material  with  a  rapidity  and  accuracy 
which  is  truly  marvelous  ;  not  to  speak  of  the  many  substances  for  which  no 
other  method  of  identification  is  known. 

"At  present  the  greatest  bar  to  its  general  use  is  the  absence  of  any  well 
denned  scheme,  and  the  absolute  necessity  of  being  well  grounded  in  general 
chemistry.  There  are  no  tables  which  can  be  followed  in  a  mechanical  way  by 
the  student,  but  on  the  contrary  he  is  obliged  to  exercise  his  knowledge  and  judg- 
ment at  every  step.  For  this  very  reason  the  introduction  of  this  subject  into  the 
list  of  those  now  taught  is  greatly  to  be  desired. ' ' 


156  MICRO-CHEMISTRY  \.CH.    VI 

It  will  be  seen  by  the  last  paragraph  that  in  chemistry  as  in 
biology,  the  microscope  is  only  an  aid  to  investigation;  it  cannot  take 
the  place  of  thorough  training  in  the  fundamentals  of  the  subject 
under  investigation. 

§  229.  The  following  list  of  substances  is  suggested  by  Dr. 
Chamot  for  beginning  practice  as  the  results  given  are  definite  and 
easily  obtained  : 

SUGGESTIONS   FOR   STUDY   ON   CRYSTAL  SYSTEMS 

"Isometric. 

Sodium  chlorid,  potassium  chlorid,  potassium  iodid.  Strontium  nitrate. 
Barium  nitrate.  Lead  nitrate.  Potassium  bromid.  Sodium  bromid. 

Alums  crystallize  in  octahedra,  cubes  or  combinations  of  the  two.  It  is 
well  to  recall  that  the  alums  have  the  general  formula,  M2(RO4)3.N2RO4.24 
H2O,  where  -M-  can  be  Al,  Cr,  Mn,  Fe,  In,  Ga,  Tl,  R  ;  -N-  Na,  K,  Rb,  Cs, 
NH4  Ag,  or  Tl  and  -R-  S  or  Se.  All  alums  are  isomorphous. 

Tetragonal. 

Potassium  copper  chlorid.     Ammonium  copper  chlorid.     Urea. 
Nickel  sulfate  6H2O.     This  salt  is  dimorphic,  crystallizing  also  in  the 
monoclinic  system.     Nickel  sulfate  yH2O  is  orthorhombic. 

Orthorhombic. 

Asparagin.     Picric  acid.     Acetanilid.     Resorcin. 

Mercuric  chlorid.     Silver  nitrate.    Potassium  sulfate.    Potassium  nitrate. 
Magnesium  sulfate  7H2O.     Potassium  chromate.     Sodium  nitrate  (also 
Hexagonal). 

Monoclinic. 

Lactose.    Napthalene.    Potassium  ferric  oxalate.     Sodium  ferric  oxalate. 

Potassium  chlorate  (sodium  chlorate  is  Isomet.  or  Tetrag.) 

Lead  acetate.     Copper  acetate  H2O.     Oxalic  acid. 

Ferrous  sulfate,  this  salt  forms  normally  with  7  H2O  and  is  then  Mono- 
clinic,  but  in  presence  of  zinc  sulfate  becomes  Orthorhombic,  and  in  presence 
of  copper  sulfate,  triclinic.  Sodium  Sulfate  ioH2O.  Borax.  Potassium  ferri- 
cyanid. 

Triclinic. 

Copper  sulfate  5H  2  O.     Boric  acid.     Potassium  dichromate. 

Hexagonal. 

Lead  iodid  (according  to  Behrens  PbI2  is  probably  orthorhombic). 
Sodium  nitrate  (also  Orthorhombic).  Bromoform.  lodoform. 

%  230.  Before  performing  analytical  tests,  it  is  necessary  that  the  student 
be  familiar  with  the  properties  of  crystals  and  also  thoroughly  familiar  with 
the  appearance  of  crystals  of  the  chlorids,  nitrates,  and  sulfates  of  Sodium, 
Potassium,  and  Ammonium  ;  since  some  of  these  salts  are  sure  to  appear  in 
almost  every  test  drop  examined. 


CH.   VI}  MICRO-CHEMISTRY  157 

Frequently  a  chemically  pure  salt  cannot  be  made  to  yield  satisfactory 
crystals  on  the  evaporation  of  its  solution,  but  beautifully  formed  crystals  will 
result  when  in  the  presence  of  other  compounds.  A  striking  example  is  found  in 
Ammonium  chlorid.  This  salt  fails  to  yield  other  than  dendritic  masses  when 
preparations  are  made  from  the  pure  salt,  but  if  formed  by  metathesis  and 
especially  if  in  the  presence  of  a  difficultly  crystallizable  salt,  well  formed  isome- 
tric crystals  (cubes)  are  seen. 

AN   EXERCISE  FOR   PRACTICE 

Take  a  fragment  of  ammonium  chlorid,  dissolve  in  a  tiny  drop  of  water  on  a 
slide  and  try  to  obtain  distinct  well  formed  crystals.  Neither  slow  nor  rapid 
evaporation  nor  recrystallization  by  breathing  on  the  preparation  will  yield  satis- 
factory crystals. 

Place  a  small  drop  of  water  on  a  glass  slide,  add  Ferric  chlorid  until  the  drop 
is  distictly  yellow.  Stir.  At  the  centre  of  the  drop  add  two  or  three  tiny  frag- 
ments of  Ammonium  acetate.  The  preparation  must  not  be  warmed.  There  is 
formed  Ferric  acetate,  Ammonium  chlorid  and  double  chlorids  of  ammonium  and 
iron.  Study  the  preparation  and  observe  the  following  points,  i.  Tendency 
toward  formation  of  double  salt.  2.  That  the  type  crystal  of  NH4C1  is  a  cube. 
3.  Cubes  may  so  grow  as  to  present  the  appearance  of  a  rectangular  prism.  4.  In 
certain  positions  cubes  have  the  appearance  of  a  pyramid.  5.  In  other  positions 
they  exhibit  a  hexagonal  outline,  thus  simulating  a  polyhedron  of  many  faces. 
6.  There  is  scarcely  any  tendency  in  this  case  toward  the  formation  of  the 
dendritic  masses  observed  in  the  first  experiment.  7.  The  crystals  often  develop 
fastest  along  the  diagonal  planes  so  that  the  regular  faces  are  replaced  by 
pyramidal  depressions. ' ' 


FIG.  127.  Czapski's  Ocular  Iris-diaphragm  with  cross 
hairs  for  examining  and  accurately  determining  the  axial  im- 
ages of  small  crystals.  The  iris  diaphragm  enables  the  observer 
to  make  the  field  as  large  or  small  as  desired. 

A.  Longitudinal  Section. 

B.  Transection,  showing  the  cross  lines  and  the  iris  dia- 
phragm with  the  projecting  part  at  the  left,  by  which  the  dia- 
phragm is  opened  and  closed.     (Zeiss*  Catalog. ) 


For  directions  and  hints  in  micro-chemical  work  and  crystallography,  consult 
the  various  volumes  of  the  Journal  of  the  Roy.  Micr.  Soc.;  Zeitschrift  fiir  physio- 
logische  Chemie,  and  other  chemical  journals  ;  Wormly  ;  Kl£ment  &  Renard  ; 
Carpenter-Dalliuger  ;  Hogg  ;  Behrens,  Kossel  und  Schiefferdecker  ;  Frey  ;  Dana', 
and  other  works  on  mineralogy  ;  Davis,  Behrens,  T.  H.— Anleitung  zur  micro- 
chemischen  Analyse  der  wichtigsten  organischen  Verbindungen.  Hamburg, 
1895-1897.  Microchemische  Technik,  2d  edition,  Hamburg,  1900.  A  manual  of 
michrochemical  analysis  with  an  introductory  chapter  by  J.  W.  Judd,  London. 


158  TEXTILE  FIBERS  \_CH.    VI 

1894.  Bspecial  attention  is  also  called  to  the  articles  of  Dr.  E.  M.  Chamot  in  the 
Journal  of  Applied  Microscopy  beginning  with  vol.  ii.  p.  502,  and  continued  in 
vol.  iii.  and  iv. 

TEXTILE   FIBERS,    FOOD    AND   PHARMACOLOGICAL   PRODUCTS 

§  231.  The  microscope  is  coming  more  and  more  into  use  for  the 
determination  of  the  character  of  textile  fibers,  both  in  the  raw  state 
and  after  manufacture.  As  the  textile  fibers  have  distinctive  char- 
acters it  is  not  difficult  to  determine  mixtures  in  fabrics  of  various 
kinds.  The  student  is  advised  to  study  carefully  known  fibers,  as  of 
cotton,  wool,  linen,  silk,  jute  etc.,  so  that  he  is  certain  of  the  appear- 
ances, and  then  to  determine  of  what  fibers  different  fabrics  are  com- 
posed. He  will  be  astonished  at  the  amount  of  "Alabama  wool"  in 
supposedly  all  wool  goods. 

For  works  and  articles  upon  textile  fibers  see  :  Herzfeld,  J.  Trans- 
lated by  Salter.  The  technical  testing  of  yarns  and  textile  fabrics 
with  reference  to  official  specifications.  London,  1898.  E.  A. 
Posselt — The  structure  of  fibers,  yarns  and  fabrics.  Philadelphia 
and  London,  1891.  Dr.  C.  Rougher — Des  filaments  vegetaux  em- 
ployes dans  Tindustrie.  Paris,  1873.  Wm.  P.  Wilson  and  E.  Fah- 
ring — ,  The  conditioning  of  wool  and  other  fabrics  in  the  technological 
laboratories  of  the  Philadelphia  Commercial  Museum.  Journal  of  Ap- 
plied Microscopy,  Vol.  II,  (1899)  pp.  290-292,  457-460.  Bulletin  of 
the  National  Association  of  Wool  Growers,  1875,  p.  470.  Proceed- 
ings of  the  Amer.  Micr.  Soc.,  1884,  PP-  65-68.  Besides  these  refer- 
ences one  is  liable  to  find  pictures  and  discussions  of  various  fibers 
in  general  works  on  the  microscope,  and  in  technical  and  general 
cyclopaedias. 

§  232.  From  the  nature  of  food  and  pharmacological  products 
adulterations  are  in  many  cases  most  accurately  and  easity  determined 
by  microscopic  examination.  The  student  will  find  constant  reference 
to  the  microscopical  characters  of  the  genuine  and  spurious  substances 
in  medicines  and  other  pharmacological  products  in  works  on  pharmacy 
or  pharmacology  ;  also  in  pharmacological  journals  and  in  druggists' 
reports,  e.  g.  the  excellent  and  well  illustrated  article  by  Burt  E. 
Nelson  of  the  chemical  laboratory  of  the  Binghamton  State  Hospital 
on  the  Microscopical  examination  of  tea,  coffee,  spices  and  condiments. 
This  appeared  in  Merck's  Report,  Oct.  15,  Dec.  15,  1898.  The  micro- 
scopical Journals  also  contain  occasional  articles  bearing  upon  this 
subject.  See  also  Food  Products  in  bulletins  of  the  U.  S.  Dep't  Agr. 
Mace,  E. — Les  substances  alimentaire,  etc.,  Paris,  1891.  Schimper, 


CH.  VI}  MICRO-METALLOGRAPHY  159 

A.  F.  W.  Anleitung,  etc.    Jena,  1900.  Hugh  Gait, — The   Microscopy 
of  the  starches,  illustrated  by  photo-micrographs,  London,  1900. 

THE   MICROSCOPE    IN  METALLOGRAPHY 

§  233.  In  the  modern  investigation  of  metals  and  alloys  much 
light  has  been  thrown  upon  the  structural  peculiarities  which  render 
some  mixtures  satisfactory  and  others  unsatisfactory.  There  are  two 
great  methods  :  First,  that  of  studying  fractured  surfaces  without  re- 
course to  any  reagents.  Second,  to  polish  a  metallic  surface  carefully 
with  emery  or  carborundum  and  finally  with  rouge  or  diamantine  and 
then  etch  it  with  some  acid  for  a  longer  or  shorter  time.  For  either 
method  reflected  light  must  be  used.  For  low  powers  that  obtained  at  a 
good  window  or  by  a  lamp  or  a  lamp  and  bulls  eye  are  good.  The  illum- 
inating objectives  (§  28),  i.  e.  objectives  in  which  a  prism  in  the  side 
of  the  objective  reflects  light  down  through  the  lenses  which  act  as  a 
condenser,  are  preferable  for  most  work  and  indeed  necessary  if  one  uses 
high  powers.  For  special  microscope  see  Fig.  126  A. 

Elaborate  arrangements  have  been  devised  for  holding  the  piece 
of  metal  on  the  stage,  but  some  beeswax,  or  some  clay  made  plastic 
with  glycerin  answers  well.  For  pictures  of  the  appearances  seen 
in  studying  metallic  surfaces,  see  the  journals  of  engineering  and 
metallurgy,  especially  the  Metallographist,  a  quarterly  publication 
devoted  to  the  study  of  metals  with  special  reference  to  their  physics 
and  micro-structure,  etc.  In  twenty-five  or  more  of  the  great  metal 
manufacturing  establishments  special  laboratories  for  microscopic 
examination  and  investigation  have  been  established.  This  is  an  illus- 
tration of  what  has  frequently  occurred — great  manufacturing  interests 
have  outrun  the  universities  in  the  appreciation  and  application  of 
methods  of  reasearch.  Fortunately,  however,  laboratories  are  already 
springing  up  in  connection  with  the  universities,  and  probably  within 
ten  years  every  great  technical  school  will  have  its  laboratory  of 
micro-metallography  where  students  will  have  opportunity  to  perfect 
themselves  in  the  preparation,  photography  and  microscopic  study  of 
the  metals  and  alloys. 

Beside  the  sources  of  information  given  above,  see  Dr.  H.  Ost  und 
Dr.  Fr.  Kolbeck,  Lehrbuch  der  chemischen  Technologic  mit  einem 
Schlussabschnitt  "  Metallurgie. "  Hannover,  1901.  Behrens,  T.  H. — 
Das  mikroskopische  Gefiige  der  Matalle,  etc.  Hamburg,  1894.  For 
an  excellent  bibliography  of  188  titles  ;  see  the  Metallographist,  Vol. 
I,  1898,  and  appended  to  the  special  papers  in  all  the  volumes.  Also 
in  Iron  Age,  Jan.  27,  1898.  Carpenter- Dallinger,  p.  264. 


i6o 


MICRO-METALLOGRAPHY 


\CH.  VI 


FIG.  126  A.  Microscope  especially  constructed  for  the  study  of  metals  and 
alloys.  (  The  Boston  Testing  Laboratories'). 

The  stage  is  rotary,  and  may  be  raised  or  lowered  by  rack  and  pinion.  Above 
the  objective  is  the  arrangement  for  illumination  (see  Ch.  VII}. 


CHAPTER  VII 


SLIDES  AND  COVER-GLASSES  ;  MOUNTING  ;  ISOLATION  ; 
SECTIONING  BY  THE  COLLODION  AND  THE  PARAF- 
FIN METHODS;  LABELING  AND  STORING  MICRO- 
SCOPICAL PREPARATIONS  ;  REAGENTS 


SLIDES    AND   COVER-GLASSES 

\  234.  Slides,  Glass  Slides  or  Slips,  Microscopic  Slides  or  Slips. — These  are 
strips  of  clear  flat  glass  upon  which  microscopic  specimens  are  usually  mounted  for 
preservation  and  ready  examination.  The  size  that  has  been  almost  universally 
adopted  for  ordinary  preparations  is  25  x  76  millimeters  (1x3  inches).  For  rock 
sections,  slides  25  x  45  mm.  or  32  x  32  mm.  are  used  ;  for  serial  sections,  slides 
25  x  76  mm.,  50  x  76  mm.  or  37  x  87  mm.  are  used.  For  special  purposes,  slides 
of  the  necessary  size  are  employed  without  regard  to  any  conventional  standard. 

Whatever  size  of  slide  is  used,  it  should  be  made  of  clear  glass  and  the  edges 
should  be  ground.  It  is  altogether  false  economy  to  mount  microscopic  objects 
on  slides  with  unground  edges.  It  is  unsafe  also  as  the  unground  edges  are  liable 
to  wound  the  hands. 

For  micro-chemical  work  Dr.  Chamot  recommends  slides  of  half  the  length  of 
those  used  in  ordinary  microscopic  work.  From  the  rapidity  with  which  they  are 
destroyed,  he  thinks  the  ground  edges  are  unnecessarily  expensive.  He  adds 
further:  "It  is  a  great  misfortune  that  the  colorless  glass  slips  used  in  America 
and  so  excellent  for  ordinary  microscopic  work  should  be  easily  attacked  by  all 
liquids  ;  even  water  extracts  a  relatively  enormous  amount  of  alkalies  and  alka- 
line earths.  The  slips  of  greenish  glass,  while  not  as  neat  or  desirable  for  general 
microscopy,  seem  to  be  decidedly  more  resistant,  and  are  therefore  preferable." 
Transparent  celluloid  slides  are  recommended  by  Behrens  for  work  where  hydro- 
fluoric acid  and  its  derivatives  are  to  be  examined.  (Chamot,  Jour.  Appl.  Micr. 
vol.  iii,  p.  793). 

\  235.  Cleaning  Slides. — For  new  slides  a  thorough  rinsing  in  clean  water  with 
subsequent  wiping  with  a  soft  cloth  like  glass  toweling,  or  thin  cotton  cloth  like 
bleached  cheese  cloth  (bunting  or  gauze,  or  absorbent  surgical  gauze),  usually  fits 
them  for  ordinary  use.  If  they  are  not  satisfactorily  cleaned  in  this  way,  soak 
them  a  short  time  in  50%  or  75%  alcohol,  let  them  drain  for  a  few  moments  on  a 
clean  towel  or  on  blotting  paper,  and  then  wipe  with  a  soft  cloth.  In  handling 
the  slides  grasp  them  by  their  edges  to  avoid  soiling  the  face  of  the  slide.  After 
the  slides  are  cleaned  they  should  be  stored  in  a  place  as  free  as  possible  from 
dust.  For  storing,  covered  glass  dishes  are  best.  Use  museum  jars  or  glass  boxes 
(Fig.  150). 


162 


SLIDES  AND  COVER-GLASSES 


\_CH.  VII 


FIG.  1 28.  Glass  slide  or  slip  of  the  ordinary  size  for  microscopic  work  ( j  x  fin., 
j6  x  25  mm.}.  ( Cut  loaned  by  the  Spencer  Lens  Company}. 

For  old  slides,  if  only  water,  glycerin  or  glycerin  jelly  has  been  used  on  them, 
they  may  be  cleaned  with  water,  or  preferably,  warm  water  and  then  with  alcohol 
if  necessary.  Where  balsam,  or  any  oily  or  gummy  substance  has  been  used  upon 
the  slides,  they  may  be  freed  from  the  balsam,  etc.,  by  soaking  them  for  a  week 
or  more  in  one  of  the  cleaning  mixtures  for  glass.  If  they  are  first  soaked  in 
xylene,  benzin  or  turpentine  to  dissolve  the  balsam,  then  soaked  in  the  cleaning 
mixture,  the  time  required  will  be  much  shortened  (\  242).  After  all  foreign 
matter  is  removed  the  slides  should  be  thoroughly  rinsed  in  water  to  remove  all 
the  cleaning  mixture.  They  may  then  be  treated  as  directed  for  new  slides. 

If  slides  with  large  covers,  as  in  mounted  series,  are  put  into  the  cleaning 
mixture,  the  swelling  of  the  balsam  is  liable  to  break  the  covers.  Dissolving 
away  the  balsam  with  turpentine,  avoids  this,  and  greatly  shortens  the  time  neces- 
sary for  cleaning  the  old  slides  and  covers. 

Another  excellent  method  for  balsam  mounts  is  to  heat  the  slides  until  the 
balsam  is  soft  and  then  remove  the  cover- glasses.  The  turpentine  cleaning  mix- 
ture, etc.,  can  then  act  on  the  entire  surface.  It  should  be  said,  however,  that  at 
the  present  price  of  slides  and  cover-glasses  it  costs  nearly  as  much  as  the  slides 
and  covers  are  worth  to  clean  those  that  have  been  used  in  balsam  mounting. 

\  236.  Cover- Glasses  or  Covering  Glasses. — These  are  circular  or  quadr- 
angular pieces  of  thin  glass  used  for  covering  and  protecting  microscopic  objects. 
They  should  be  very  thin,  o.io  to  0.25  millimeter  (see  table,  \  29).  It  is  better 
never  to  use  a  cover-glass  over  0.20  mm.  thick,  then  the  preparation  may  be  studied 
with  a  2  mm.  oil  immersion  as  well  as  with  lower  objectives.  Except  for  objects 
wholly  unsuited  for  high  powers,  it  is  a  great  mistake  to  use  cover-glasses  thicker 
than  the  working  distance  of  a  homogeneous  objective  (\  61).  Indeed,  if  one 
wishes  to  employ  high  powers,  the  thicker  the  sections  the  thinner  should  be  the 
cover-glass  (see  $  240). 

The  cover-glass  should  always  be  considerably  larger  than  the  object  ovei 
which  it  is  placed. 

\  237.  Cleaning  Cover-Glasses. — New  cover-glasses  should  be  put  into  a 
glass  dish  of  some  kind  containing  one  of  the  cleaning  mixtures  ($  242)  and  al- 
lowed to  remain  a  day  or  longer.  In  putting  them  in,  push  one  in  at  a  time  and 
be  sure  that  each  is  entirely  immersed,  otherwise  they  adhere  very  closely  and  the 
cleaning  mixture  is  unable  to  act  freely.  Soiled  covers  should  be  left  a  week  or 
more  in  the  cleaning  mixture.  An  indefinite  sojourn  in  the  cleaner  does  not  seem 


CH.  VII]  SLIDES  AND  COVER-GLASSES  163 

to  injure  the  slides  or  covers.  After  one  day  or  longer,  pour  off  the  cleaning  mix- 
ture into  another  glass  jar,  and  rinse  the  cover-glasses,  moving  them  around  with 
a  gentle  rotary  motion.  Continue  the  rinsing  until  all  the  cleaning  mixture  is 
removed.  One  may  rinse  them  occasionally,  and  in  the  meantime  allow  a  very 
gentle  stream  of  water  to  flow  on  them,  or  they  may  be  allowed  to  stand  quietly 
and  have  the  water  renewed  from  time  to  time.  When  the  cleaning  mixture  is 
removed  rinse  the  covers  well  with  distilled  water,  and  then  cover  them  with  50% 
to  75%  alcohol. 


FIGS.  129-130.  Figures  of  square  and  of  circular  cover-glasses.  See  also  Fig. 
162  for  covers  on  serial  sections. 

\  238.  Wiping  the  Cover- Glasses. — When  ready  to  wipe  the  cover-glasses, 
remove  several  from  the  alcohol  and  put  them  on  a  soft,  dry  cloth,  or  on  some  of 
the  lens  or  filter  paper  to  let  them  drain.  Grasp  a  cover-glass  by  its  edges,  cover 
the  thumb  and  index  finger  of  the  other  hand  with  a  soft,  clean  cloth  or  some  of  the 
the  lens  paper.  The  bleached  cheese  cloth  ( \  235 )  is  good  for  wiping  covers.  Grasp 
the  cover  between  the  thumb  and  index  and  rub  the  surfaces.  In  doing  this  it  is 
necessary  to  keep  the  thumb  and  index  well  opposed  on  directly  opposite  faces  of 
the  cover  so  that  no  strain  will  come  on  it,  otherwise  the  cover  is  liable  to  be 
broken. 

When  a  cover  is  well  wiped,  hold  it  up  and  look  through  it  toward  some  dark 
object.  The  cover  will  be  seen  partly  by  transmitted  and  partly  by  reflected  light, 
and  any  cloudiness  will  be  easily  detected.  If  the  cover  does  not  look  clear,  breathe 
on  the  faces  and  wipe  again.  If  it  is  not  possible  to  get  a  cover  clear  in  this  way 
it  should  be  put  again  into  the  cleaning  mixture. 

As  the  covers  are  wiped  put  them  in  a  clean  glass  box  or  Petri  dish.  Handle 
them  always  by  their  edges,  or  use  fine  forceps.  Do  not  put  the  fingers  on  the 
faces  of  the  covers,  for  that  will  surely  cloud  them. 

FIG.  131.  Glass  dish  for  holding  covers 
(  Whitall,  Tatum  &  Co.}. 

\  239.  Cleaning  Large  Cover-Glasses. — For 
serial  sections  and  especially  large  sections, 
large  quadrangular  covers  are  used  (Fig.  162). 
These  are  to  be  put  one  by  one  into  a  cleaning 
mixture  as  for  the  smaller  covers  and  treated  in 

every  way  the  same.  In  wiping  them  one  may  proceed  as  for  the  small  covers, 
but  special  care  is  necessary  to  avoid  breaking  them.  It  is  especially  desirable 
that  these  large  covers  should  be  thin — not  over  0.15-0.20  mm.  otherwise  high  ob- 
jectives cannot  be  used  in  studying  the  preparations. 

$  240.  Measuring  the  Thickness  of  Cover-Glasses. — It  is  of  the  greatest 
advantage  to  know  the  exact  thickness  of  the  cover-glass  on  an  object  ;  for,  (a) 


164 


SLIDES  AND  COVER-GLASSES 


VII 


In  studying  the  preparation  one  would  not  try  to  use  objectives  of  a  shorter  work- 
ing distance  than  the  thickness  of  the  cover  (§61);  (b)  In  using  adjustable 
objectives  with  the  collar  graduated  for  different  thicknesses  of  cover,  the  collar 
might  be  set  at  a  favorable  point  without  loss  of  time  ;  (c)  For  unadjustable 
objectives  the  thickness  of  cover  may  be  selected  corresponding  to  that  for  which 
the  objective  was  corrected  (see  table,  \  29).  Furthermore,  if  there  is  a  variation 
from  the  standard,  one  may  remedy  it,  in  part  at  least,  by  lengthening  the  tube  if 
the  cover  is  thinner,  and  shortening  it  if  the  cover  is  thicker  than  the  standard 
(\  102,  Fig.  133.) 

In  the  so  called  No.  i  cover-glasses  of  the  dealers  in  microscopical  supplies, 
the  writer  has  found  covers  varying  from  o.  10  mm.  to  0.35  mm.  To  use  cover- 
glasses  of  so  wide  a  variation  in  thickness  without  knowing  whether  one  has  a 
thick  or  thin  one  is  simply  to  ignore  the  fundamental  principles  by  which  correct 
microscopic  images  are  obtained. 


32nds. 
1    .0312 
3  .0.937 
5  .1562 
7 .2187 
9  .2812 
11.3437 
13.4062 
15.4687 


FIG.  132.  Micrometer  Calipers  (Brown  and  Sharpe}.  Pocket  Calipers,  gradu- 
ated in  inches  or  millimeters,  and  well  adapted  for  measuring  cover-glasses. 

It  is  then  strongly  recommended  that  every  preparation  shall  be  covered  with 
a  cover-glass  whose  thickness  is  known,  and  that  this  thickness  should  be  indicated 
in  some  way  on  the  preparation. 

|  241.  Cover-Glass  Measurers,  Testers  or  Gauges.  For  the  purpose  of 
measuring  cover-glasses  there  are  three  very  excellent  pieces  of  apparatus.  The 
micrometer  calipers  (Fig.  132)  used  chiefly  in  the  mechanic  arts,  are  convenient 
and  from  their  size  easily  carried  in  the  pocket.  The  two  cover-glass  measurers 
specially  designed  for  the  purpose  are  shown  in  Figs.  133-134.  With  either  of 
these  the  covers  may  be  more  rapidly  measured  than  with  the  calipers. 

With  all  of  these  measures  or  gauges  one  should  be  certain  that  the  index 
stands  at  zero  when  at  rest.  If  the  index  does  not  stand  at  zero  it  should  be 
adjusted  to  that  point,  otherwise  the  readings  will  not  be  correct. 

As  the  covers  are  measured  the  different  thicknesses  should  be  put  into 
different  boxes  and  properly  labeled.  Unless  one  is  striving  for  the  most  accurate 
possible  results,  cover-glasses  not  varying  more  than  0.06  mm.  may  be  put  in  the 
same  box.  For  example,  if  one  takes  0.15  mm.  as  a  standard,  covers  varying  0.03 
mm.  on  each  side  may  be  put  into  the  same  box.  In  this  case  the  box  would  con- 
tain covers  of  0.12,  0.13,  0.14,  0.15,  0.16,  0.17  and  o.iS  mm. 


CH.   F/7] 


SLIDES  AND  COVER-GLASSES 


FIG.  133.     Cover-Glass  Measurer  (Edward  Bausch}. 

The  cover  glass  is  placed  in  the  notch  between  the  two  screws,  and  the  drum  is 
turned  by  the  milled  head  at  the  right  till  the  cover  is  in  contact  with  the  screws. 
The  thickness  is  then  indicated  by  the  knife  edge  on  the  drum  and  may  be  read  off 
directly  in  o.ooi  of  a  millimeter  or  inch.  In  other  columns  is  given  the  proper 
tube-length  for  various  unadjustable  objectives  (\,  i,  \,  and  ^  in. )  (Bausch  and 
Lomb  Optical  Company}. 


FIG.  134.  Zeiss  Cover-Glass 
Measu  rer.  With  th  is  the  knife  edge 
jaws  are  opened  by  means  of  a  lever 
and  the  cover  inserted.  The  thick- 
ness may  then  be  read  off  on  the  face 
as  the  pointer  indicates  the  thick- 
ness in  hundredths  millimeter  in 
the  outer  circle  and  in  thousandths 
inch  on  the  inner  circle. 


\  242.  Cleaning  Mixtures  for  Glass. — The  cleaning  mixtures  used  for  clean- 
ing slides  and  cover-glasses  are  those  commonly  used  in  chemical  laboratories : 
(Dr.  G.  C.  Caldwell's  Laboratory  Guide  in  Chemistry). 


1 66  MOUNTING  AND  LABELING  [CH.  VII 

(A)  Bichromate  of  Potash  and  Sulphuric  Acid. 

Bichromate  of  potash  (K2Cr2O7)  200  grams 

Water,  distilled  or  ordinary  -       800  cc. 

Sulphuric  acid  (H2SO4)  1200  cc. 

Dissolve  the  dichromate  in  the  water  by  the  aid  of  heat,  using  an  agate  or 
other  metal  dish,  then  pour  it  into  a  heavy  iron  kettle  lined  with  sheet  lead  (Pr. 
Trans.  Amer.  Micr.  Soc.,  1899,  p.  107).  Add  the  sulphuric  acid  to  the  dissolved 
dichromate  in  the  kettle.  The  purpose  of  the  lead  lined  kettle  is  to  avoid  break- 
age from  the  great  heat  developed  upon  the  addition  of  the  sulphuric  acid.  The 
lead  is  very  slightly  affected  by  the  acid,  iron  would  be  corroded  by  it. 

For  making  this  mixture,  ordinary  water,  commercial  dichromate  and  strong 
commercial  sulphuric  acid  may  be  used.  It  is  not  necessary  to  employ  chemically 
pure  materials. 

This  is  an  excellent  cleaning  mixture  and  is  practically  odorless.  It  is  exceed- 
ingly corrosive  and  must  be  kept  in  glass  vessels.  It  may  be  used  more  than 
once,  but  when  the'  color  changes  markedly  from  that  seen  in  the  fresh  mixture  it 
should  be  thrown  away. 

( B )  Sulphuric  and  Nitric  Acid  Mixture. 

Nitric  acid  (HNO3)  200  cc. 

Sulphuric  acid  (H^SO^)  -        300  cc. 

The  acids  should  be  strong,  but  they  need  not  be  chemically  pure.  The  two 
acids  are  mixed  slowly,  and  kept  in  a  glass  stoppered  bottle.  This  is  a  more  cor- 
rosive mixture  than  (A),  and  has  the  undesirable  feature  of  giving  off  stifling 
fumes,  therefore  it  must  be  carefully  covered.  It  may  be  used  several  times.  It 
acts  more  rapidly  than  the  dichromate  mixture,  but  on  account  of  the  fumes  is  not 
so  well  adapted  for  general  laboratories. 

MOUNTING,    AND   PERMANENT    PREPARATION   OF   MICROSCOPICAL   OBJECTS 

\  243.  Mounting  a  Microscopical  Object  is  so  arranging  it  upon  some  suit- 
able support  (glass  slide)  and  in  some  suitable  mounting  medium  that  it  may  be 
satisfactorily  studied  with  the  microscope. 

The  cover-glass  on  a  permanent  preparation  should  always  be  considerably 
larger  than  the  object ;  and  where  several  objects  are  put  under  one  cover- glass  it  is 
false  economy  to  crowd  them  too  closely  together. 

\  244.  Temporary  Mounting. — In  a  great  many  cases  objects  do  not  need  to 
be  preserved  ;  they  are  then  mounted  in  any  way  to  enable  one  best  to  study 
them,  and  after  the  study  the  cover  glass  is  removed,  the  slide  cleaned  and  made 
ready  for  future  use.  In  the  study  of  living  objects,  of  course  only  temporary 
preparations  are  possible.  With  amoebae,  white  blood  corpuscles,  and  many 
other  objects  both  animal  and  vegetable,  the  living  phenomena  can  best  be  studied 
by  mounting  them  in  the  natural  medium.  That  is,  for  amoebae,  in  the  water  in 
which  they  are  found  ;  for  the  white  blood  corpuscles,  a  drop  of  blood  is  used  and, 
as  the  blood  soon  coagulates,  they  are  in  the  serum.  Sometimes  it  is  not  easy  or 
convenient  to  get  the  natural  medium,  then  some  liquid  that  has  been  found  to 
serve  in  place  of  the  natural  medium  is  used.  For  many  things,  water  with  a 


CH. 


MOUNTING  AND  LABELING 


167 


little  common  salt  (water  100  cc.,  common  salt  T%ths  gram)  is  employed.  This  is 
the  so-called  normal  salt  or  saline  solution.  For  the  ciliated  cells  from  frogs  and 
other  amphibia,  nothing  has  been  found  so  good  as  human  spittle.  Whatever  is 
used,  the  object  is  put  on  the  middle  of  the  slide  and  a  drop  of  the  mounting 
medium  added,  and  then  the  cover-glass.  The  cover  is  best  put 
on  with  fine  forceps,  as  shown  in  Fig.  136.  After  the  cover  is  in 
place,  if  the  preparation  is  to  be  studied  for  some  time,  it  is  better 
to  avoid  currents  and  evaporation  by  painting  a  ring  of  castor  oil 
around  the  cover  in  such  a  way  that  part  of  the  ring  will  be  on  the 
slide  and  part  on  the  cover  (Fig.  165. ) 


FIG.  135.  Needle  Holder  (Queen  &  Co.}.  By  means  of  the 
screw  clamp  or  chuck  at  one  end  the  needle  may  be  quickly 
changed. 

FIG.  136.  To  show  the  method  of  putting  a  cover-glass  upon  a 
microscopic  preparation.  The  cover  is  grasped  by  one  edge,  the 
opposite  edge  is  then  brought  down  to  the  slide,  and  the  cover 
gradually  lowered  upon  the  object. 

FIG.  136. 

§  245.  Permanent  Mounting. — For  making  permanent  microscopical  prepara- 
tions, there  are  three  great  methods.  Special  methods  of  procedure  are  necessary 
to  mount  objects  successfully  in  each  of  these  ways.  The  best  mounting  medium 
and  the  best  method  of  mounting  in  a  given  case  can  only  be  determined  by  ex- 
periment. In  most  cases  some  previous  observer  has  already  made  the  necessary 
experiments  and  furnished  the  desired  information. 

The  three  methods  are  the  following  :  ( A)  Dry  or  in  air  (g  246);  (B)  In  some 
medium  miscible  with  water,  as  glycerin  or  glycerin  jelly  (|  250);  (C)  In  some 
resinous  medium  like  Canada  Balsam  (§  255). 

§  246.  Mounting  Dry  or  in  Air. — The  object  should  be  thoroughly  dry.  If 
any  moisture  remains  it  is  liable  to  cloud  the  cover-glass,  and  the  specimen  may 
deteriorate.  As  the  specimen  must  be  sealed,  it  is  necessary  to  prepare  a  cell 
slightly  deeper  than  the  object  is  thick.  This  is  to  support  the  cover-glass,  and 
also  to  prevent  the  running  in  by  capillarity  of  the  sealing  mixture. 

\  2463.     Order  of  Procedure  in  Mounting  Objects  Dry  or  in  Air. 

1.  A  cell  of  some  kind  is  prepared.     It  should  be  slightly  deeper  than  the 
object  is  thick  (§  248). 

2.  The  object  is  thoroughly  dried  (dessicated)  either  in  dry  air  or  by  the  aid 
of  gentle  heat. 

3.  If  practicable  the  object  is  mounted  on  the  cover-glass  ;  if  not  it  is  placed 
in  the  bottom  of  the  cell. 

4.  The  slide  is  warmed  till  the  cement  forming  the  cell  wall  is  somewhat 
sticky,  or  a  very  thin  coat  of  fresh  cement  is  added  ;  the  cover  is  warmed  and  put 


1 68  MOUNTING  AND  LABELING  \_CH.  VII 

on  the  cell  and  pressed  down  all  around  till  a  shining  ring  indicates  its  adherence 

(2  249)- 

5.  The  cover-glass  is  sealed  (\  249). 

6.  The  slide  is  labeled  (|  308). 

7.  The  preparation  is  cataloged  and  safely  stored  (\  309,  311). 

§  247.  Example  of  Mounting  Dry,  or  in  Air. — Prepare  a  shallow  cell  and  dry 
it  (§  248).  Select  a  clean  cover-glass  slightly  larger  than  the  cell.  Pour  upon  the 
cover  a  drop  of  10%  solution  of  salycilic  acid  in  95%  alcohol.  Let  it  dry  spon- 
taneously. Warm  the  slide  till  the  cement  ring  or  cell  is  somewhat  sticky,  then 
warm  the  cover  gently  and  put  it  on  the  cell,  crystals  down.  Press  on  the  cover 
all  around  the  edge  (§  246);  seal,  label  and  catalog  (\  253,  308,  309).  • 

A  preparation  of  mammalian  red  blood  corpuscles  may  be  satisfactorily  made 
by  spreading  a  very  thin  layer  of  fresh  blood  on  a  cover  with  the  end  of  a  slide. 
After  it  is  dry,  warm  gently  to  remove  the  last  traces  of  moisture  and  mount  blood 
side  down,  precisely  as  for  the  crystals.  One  can  get  the  blood  as  directed  for  the 
Micro-spectroscopic  work  (g  210). 


FIG.  137.  Turn-Table  for  sealing  cover-glasses  and  making  shallow  mount- 
ing cells.  ( Queen  &  Co.) 

|  248.  Preparation  of  Mouuting  Cells. — (A)  Thin  Cells.  These  are  most 
conveniently  made  of  some  of  the  cements  used  in  microscopy.  Shellac  is  one  of 
the  best  and  most  generally  applicable.  To  prepare  a  shellac  cell  place  the  slide 
on  a  turn-table  (Fig.  137)  and  center  it,  that  is,  get  the  center  of  the  slide  over 
the  center  of  the  turn-table.  Select  a  guide  ring  on  the  turn-table  which  is  a  little 
smaller  than  the  cover-glass  to  be  used,  take  the  brush  from  the  shellac,  being 
sure  that  there  is  not  enough  cement  adhering  to  it  to  drop.  Whirl  the  turn-table 
and  hold  the  brush  lightly  on  the  slide  just  over  the  guide  ring  selected.  An 
even  ring  of  the  cement  should  result.  If  it  is  uneven,  the  cement  is  too  thick  or 
too  thin,  or  too  much  was  on  the  brush.  After  a  ring  is  thus  prepared  remove 
the  slide  and  allow  the  cement  to  dry  spontaneously,  or  heat  the  slide  in  some 
way.  Before  the  slide  is  used  for  mounting,  the  cement  should  be  so  dry  when  it 
is  cold  that  it  does  not  dent  when  the  finger  nail  is  applied  to  it. 

A  cell  of  considerable  depth  may  be  made  with  the  shellac  by  adding  succes- 
sive layers  as  the  previous  one  drys. 

(B)  Deep  Cells  are  sometimes  made  by  building  up  cement  cells,  but  more 
frequently,  paper,  wax,  glass,  hard  rubber,  or  some  metal  is  used  for  the  main 
part  of  the  cell.  Paper  rings,  block  tin  or  lead  rings  are  easily  cut  out  with  gun 
punches.  These  rings  are  fastened  to  the  slide  by  using  some  cement  like  the 
shellac. 


CH.  VII] 


MOUNTING  AND  LABELING 


169 


|  249.  Sealing  the  Cover-Glass  for  Dry  Objects  Mounted  in  Cells. — When 
an  object  is  mounted  in  a  cell,  the  slide  is  warmed  until  the  cement  is  slightly 
sticky  or  a  very  thin  coat  of  fresh  cement  is  put  on.  The  cover-glass  is  warmed 
slightly  also,  both  to  make  it  stick  to  the  cell  more  easily,  and  to  expel  any  re- 
maining moisture  from  the  object.  When  the  cover  is  put  on  it  is  pressed  down 
all  around  over  the  cell  until  a  shining  ring  appears,  showing  that  there  is  an  in- 
timate contact.  In  doing  this  use  the  convex  part  of  the  fine  forceps  or  some 
other  blunt,  smooth  object  ;  it  is  also  necessary  to  avoid  pressing  on  the  cover 
except  immediately  over  the  wall  of  the  cell  for  fear  of  breaking  the  cover.  When 
the  cover  is  in  contact  with  the  wall  of  cement  all  around,  the  slide  should  be 
placed  on  the  turn-table  and  carefully  arranged  so  that  the  cover-glass  and  cell 
wall  will  be  concentric  with  the  guide  rings  of  the  turn-table.  Then  the  turn- 
table is  whirled  and  a  ring  of  fresh  cement  it  painted,  half  on  the  cover  and  half 
on  the  cell  wall  (Fig.  165.)  If  the  cover-glass  is  not  in  contact  with  the  cell  wall 
at  any  point  and  the  cell  is  shallow,  there  will  be  great  danger  of  the  fresh  cement 
running  into  the  cell  and  injuring  or  spoiling  the  preparation.  When  the  cover- 
glass  is  properly  sealed,  the  preparation  is  put  in  a  safe  place  for  the  drying  of  the 
cement.  It  is  advisable  to  add  a  fresh  coat  of  cement  occasionally. 


FIG.  138.  Centering  Card.  A  card  with  stops  for  the  slide  and  circles  in  the 
position  occupied  by  the  center  of  the  slide.  If  the  slide  is  put  upon  such  a  card  it 
is  very  easy  to  arrange  the  object  so  that  it  will  be  approximately  in  the  center  oj 
the  slide.  The  position  of  the  long  cover  used  for  serial  sections  is  also  shown 
(Fig.  162}.  (From  the  Microscope,  December,  1886). 

§  250.  Mounting  Objects  in  Media  Miscible  with  Water. — Many  objects  are 
so  greatly  modified  by  drying  that  they  must  be  mounted  in  some  medium  other 
than  air.  In  some  cases  water  with  something  in  solution  is  used.  Glycerin  of 
various  strengths,  and  glycerin  jelly  are  also  much  employed.  All  these  media 
keep  the  object  moist  and  therefore  in  a  condition  resembling  the  natural  one. 
The  object  is  usually  and  properly  treated  with  gradually  increasing  strengths  of 
glycerin  or  fixed  by  some  fixing  agent  before  being  permanently  mounted  in 
strong  glycerin  or  either  of  the  other  media. 


170  MOUNTING  AND  LABELING  [CH.  VII 

In  all  of  these  different  methods,  unless  glycerin  of  increasing  strengths  has 
been  used  to  prepare  the  tissue,  the  fixing  agent  is  washed  away  with  water  before 
the  object  is  finally  and  permanently  mounted  in  either  of  the  media. 

For  glycerin  jelly  no  cell  is  necessary  unless  the  object  has  a  considerable 
thickness. 

$  251.     Order  of  Procedure  in  Mounting  Objects  in  Glycerin. 

1.  A  cell  must  be  prepared  on  the  slide  if  the  object  is  of  considerable  thick- 
ness (§  248,  249). 

2.  A  suitably  prepared  object  ( §  250)  is  placed  on  the  center  of  a  clean  slide, 
and  if   no   cell   is   required   a  centering  card  is  used  to  facilitate  the  centering 

(Fig.  138)- 

3.  A  drop  of  pure  glycerin  is  put  upon  the  object,  or  if  a  cell  is  used,  enough 
to  fill  the  cell. 

4.  In  putting  on  the  cover-glass  it  is  grasped  with  fine  forceps  and  the  under 
side  breathed  on  to  slightly  moisten  it  so  that  the  glycerin  will  adhere,  then  one 
edge  of  the  cover  is  put  on  the  cell  or  slide  and  the  cover  gradually  lowered  upon 
the  object  (Fig.  136).     The  cover  is  then  gently  pressed  down.     If  a  cell  is  used,  a 
a  fresh  coat  of  cement  is  added  before  mounting  ($  249). 

FIG.  139.  Slide  and  cover-glass  showing  method  of 
anchoring  a  cover-glass  with  a  glycerin  preparation  when 
no  cell  is  used.  A  cover-glass  so  anchored  is  not  liable  to 
move  when  the  cover  is  being  sealed  ($  253). 

FIG.  140.  Glass  slide  with  cover-glass,  a  drop  of  re- 
agent and  a  bit  of  absorbent  paper  to  show  method  of  irri- 
gation (\262,  263}. 

5.  The  cover-glass  is  sealed  (\  249). 

6.  The  slide  is  labeled  ( \  308). 

7.  The  preparation  is  cataloged  and  safely  stored  (§  309,  311). 

$  252.     Order  of  Procedure  in  Mounting  Objects  in  Glycerin  Jelly. 

1.  Unless  the  object  is  quite  thick  no  cell  is  necessary  with  glycerin  jelly. 

2.  A  slide  is  gently  warmed  and  placed  on  the  centering  card  (Fig.  138)  and 
a  drop  of  warmed  glycerin  jelly  is  put  on  its  center.     The  suitably  prepared  object 
is  then  arranged  in  the  center  of  the  slide. 

3.  A  drop  of  the  warm  glycerin  jelly  is  then  put  on  the  object,  or  if  a  cell  is 
used  it  is  filled  with  the  medium. 

4.  The  cover-glass  is  grasped   with  fine  forceps,  the  lower  side  breathed  on 
and  then  gradually  lowered  upon  the  object  (Fig.  136)  and  gently  pressed  down. 

5.  After  mounting,  the  preparation   is   left  flat   in   some  cool  place  till  the 
glycerin  jelly  sets,  then  the  superfluous  amount  is  scraped  and  wiped  away  and 
the  cover-glass  sealed  with  shellac  ($  253). 

6.  The  slide  is  labeled  (§308). 

7.  The  preparation  is  cataloged  and  safely  stored  (\  309,  311). 

\  253.  Sealing  the  Cover-Glass  when  no  Cell  is  used. — (A)  For  glycerin 
mounted  specimens.  The  superfluous  glycerin  is  wiped  away  as  carefully  as  possi- 
ble with  a  moist  cloth,  then  four  minute  drops  of  cement  are  placed  at  the  edge  of 
the  cover  (Fig.  139),  and  allowed  to  harden  for  half  an  hour  or  more.  These  will 


CH.  VII] 


MOUNTING  AND  LABELING 


171 


anchor  the  cover-glass,  then  the  preparation  may  be  put  on  the  turn-table  and  a 
ring  of  cement  put  around  the  edge  while  whirling  the  turn-table. 

c 


FIG.  141.  A — Simple  form  of  moist  chamber  made  with  a  plate  and  bowl.  B, 
bowl  serving  as  a  bell  jar  ;  P,  plate  containing  the  water  and  over  which  the  bowl  is 
inverted  ;  S,  slides  on  which  are  mounted  preparations  which  are  to  be  kept  moist. 
These  slides  are  seen  endwise  and  rest  upon  a  bench  made  by  cementing  short  pieces 
of  large  glass  tubing  to  a  strip  of  glass  of  the  desired  length  and  width. 

B — Two  covet -glasses  (C)  made  eccentric,  so  that  they  may  be  more  easily  sepa- 
rated by  grasping  the  projecting  edge. 

C — Slide  (S)  with  projecting  cover-glass  (C).  The  projection  of  the  cover  en- 
ables one  to  grasp  and  raise  it  without  danger  of  moving  it  on  the  slide  and  thus 
folding  the  substance  under  the  cover.  (From  Proc.  Amer.  Micr.  Soc. ,  1891). 

(B)  For  objects  in  glycerin  jelly,  Farrants*  solution  or  a  resinous  medium. 
The  mounting  medium  is  first  allowed  to  harden,  then  the  superfluous  medium  is 
scraped  away  as  much  as  possible  with  a  knife,  and  then  removed  with  a  cloth 
moistened  with  water  for  the  glycerin  jelly  and  Farrants'  solution  or  with  alcohol, 
chloroform  or  turpentine,  etc.,  if  a  resinous  medium  is  used.  Then  the  slide  is  put 
on  a  turn-table  and  a  ring  of  the  shellac  cement  added.  ( C )  Balsam  preparations 
may  be  sealed  with  shellac  as  soon  as  they  are  prepared,  but  it  is  better  to  allow 
them  to  dry  for  a  few  days.  One  should  never  use  a  cement  for  sealing  prepara- 
tions in  balsam  or  other  resinous  media  if  the  solvent  of  the  cement  is  a  solvent 
also  of  the  balsam,  etc.  Otherwise  the  cement  will  soften  the  balsam  and  finally 
run  in  and  mix  with  it,  and  partly  or  wholly  ruin  the  preparation.  Shellac  is 
an  excellent  cement  for  sealing  balsam  perparations,  as  it  never  runs  in.  Balsam 
preparations  are  rarely  sealed. 

|  254.  Example  of  Mounting  in  Glycerin  Jelly. — For  this  select  some  stained 
and  isolated  muscular  fibres  or  other  suitably  prepared  objects.  (See  under  isola- 
tion §  259).  Arrange  them  on  the  middle  of  a  slide,  using  the  centering  card,  and 
mount  in  glycerin  jelly  as  directed  in  \  252.  Air  bubbles  are  not  easily  removed 
from  glycerin  jelly  preparations,  so  care  should  be  taken  to  avoid  them. 

\  255.  Mounting  Objects  in  Resinous  Media. — While  the  media  miscible 
with  water  offer  many  advantages  for  mounting  animal  and  vegetable  tissues  the 
preparations  so  made  are  liable  to  deteriorate.  In  many  cases,  also,  they  do  not 
produce  sufficient  transparency  to  enable  one  to  use  high  enough  powers  for  the 
demonstration  of  minute  details. 


172 


MOUNTING  AND  LABELING 


[CH.  VII 


By  using  sufficient  care  almost  any  tissue  may  be  mounted  in  a  resinous 
medium  and  retain  all  its  details  of  structure. 

For  the  successful  mounting  of  an  object  in  a  resinous  medium  it  must  in 
some  way  be  deprived  of  all  water  and  all  liquids  not  miscible  with  the  resinous 
mounting  medium.  There  are  two  methods  of  bringing  this  about  :  (A)  By  dry- 
ing or  desiccation  (\  256),  and  (B)  by  successive  displacements  (g  258). 


FIG.  142.  Small spirit  lamp  modified  into  a  balsam 
bottle,  a  glycerin  or  glycerin  -jelly  bottle,  or  a  bottle 
for  homogeneous  immersion  liquid.  For  all  of  these 
purposes  it  should  contain  a  glass  rod  as  shown  in  the 
figure.  By  adding  a  small  brush,  it  answers  well  for 
a  shellac  bottle  also  (See  Fig.  170) . 


\  256.  Order  of  Procedure  in  Mounting  Objects  in  Resinous  Media  by 
Desiccation  : 

1.  The  object  suitable  for  the  purpose  (fly's  wings,  etc. )  is  thoroughly  dried 
in  dry  air  or  by  gentle  heat. 

2.  The  object  is  arranged  as  desired  in  the  center  of  a  clean  slide  on  the 
centering  card  (Fig.  138). 

3.  A  drop  of  the  mounting  medium  is  put  directly  upon  the  object  or  spread 
on  a  cover-glass. 

4.  The  cover-glass  is  put  on  the  specimen  with  fine  forceps  (Fig.  136),  but  in 
no  case  does  one  breathe  on  the  cover  as  when  media-miscible  with  water  are 
used. 

5.  The  cover-glass  is  pressed  down  gently. 

6.  The  slide  is  labeled  (§308). 

7.  The  preparation  is  cataloged  and  safely  stored  (\  309,  311). 

|  257.  Example  of  Mounting  in  Balsam  by  Desiccation. — Find  a  fresh  fly, 
or  if  in  winter,  procure  a  dead  one  from  a  window  sill  or  a  spider's  web. 
Remove  the  fly's  wings,  being  especially  careful  to  keep  them  the  dorsal  side 
up.  With  a  camel's  hair  brush  remove  any  dirt  that  may  be  clinging  to  them. 
Place  a  clean  slide  on  the  centering  card,  then  with  fine  forceps  put  the  two  wings 
within  one  of  the  guide  rings.  Leave  one  dorsal  side  up,  turn  the  other  ventral 
side  up.  Spread  some  Canada  balsam  on  the  face  of  the  cover-glass  and  with  the 
fine  forceps  place  the  cover  upon  the  wings  (Fig.  136).  Probably  some  air-bubbles 
will  appear  in  the  preparation,  but  if  the  slide  is  put  in  a  warm  place  these  will 
soon  disappear.  Label,  catalog,  etc.,  ( $307-31 1). 

§  258.  Mounting  in  Resinous  Media  by  a  Series  of  Displacements. — For  ex- 
amples of  this  see  the  procedure  in  the  paraffin  and  in  the  collodion  methods 


CH.  VII]  ISOLATION  OF  ELEMENTS  173 

($  280,  300).  The  first  step  in  the  series  is  Dehydration,  that  is,  the  water  is  dis- 
placed by  some  liquid  which  is  miscible  both  with  the  water  and  the  next  liquid 
to  be  used.  Strong  alcohol  (95%  or  stronger)  is  usually  employed  for  this.  Plenty 
of  it  must  be  used  to  displace  the  last  trace  of  water.  The  tissue  may  be  soaked 
in  a  dish  of  the  alcohol,  or  alcohol  from  a  pipette  may  be  poured  upon  it.  Dehy- 
dration usually  occurs  in  the  thin  objects  to  be  mounted  in  balsam  in  5  to  15  min- 
utes. If  a  dish  of  alcohol  is  used  it  must  not  be  used  too  many  times,  as  it  loses 
in  strength. 

The  second  step  is  clearing.  That  is,  some  liquid  which  is  miscible  with  the 
alcohol  and  also  with  the  resinous  medium  is  used.  This  liquid  is  highly  refrac- 
tive in  most  cases,  and  consequently  this  step  is  called  clearing  and  the  liquid  a 
clearer.  The  clearer  displaces  the  alcohol,  and  renders  the  object  more  or  less 
translucent.  In  case  the  water  was  not  all  removed,  a  cloudiness  will  appear  in 
parts  or  over  the  whole  of  the  preparation.  In  this  case  the  preparation  must  be 
returned  to  alcohol  to  complete  the  dehydration. 

One  can  tell  when  a  specimen  is  properly  cleared  by  holding  it  over  some 
dark  object.  If  it  is  cleared  it  can  be  seen  only  with  difficulty,  as  but  little  light 
is  reflected  from  it.  If  it  is  held  toward  the  window,  however,  it  will  appear 
translucent. 

The  third  and  final  step  is  the  displacement  of  the  clearer  by  the  resinous 
mounting  medium. 

The  specimen  is  drained  of  clearer  and  allowed  to  stand  for  a  short  time  till 
there  appears  the  first  sign  of  dullness  from  evaporation  of  the  clearer  from  the 
surface.  Then  a  drop  of  the  resinous  medium  is  put  on  the  object,  and  finally  a 
cover-glass  is  placed  over  it,  or  a  drop  of  the  mounting  medium  is  spread  on  the 
cover  and  it  is  then  put  on  the  object. 

ISOLATION   OF  HISTOLOGICAI,   ELEMENTS 

|  259.  For  a  correct  conception  of  the  forms  of  the  cells  and  fibers  of  the 
various  organs  of  the  body,  one  must  see  these  elements  isolated  and  thus  be  able 
to  inspect  them  from  all  sides.  It  frequently  occurs  also  that  the  isolation  is  not 
quite  complete,  and  one  can  see  in  the  clearest  manner  the  relations  of  the  cells 
or  fibers  to  one  another. 

The  chemical  agents  or  solutions  for  isolating  are,  in  general,  the  same  as 
those  used  for  hardening  and  fixing.  But  the  solutions  are  only  about  one-tenth 
as  strong  as  for  fixing,  and  the  action  is  very  much  shorter,  that  is,  from  one  or 
two  hours  to  as  many  days.  In  the  weak  solution  the  cell  cement  or  connective 
tissue  is  softened  so  that  the  cells  and  fibers  may  be  separated  from  one  another, 
and  at  the  same  time  the  cells  are  preserved.  In  fixing  and  hardening,  on  the 
other  hand,  the  cell  cement,  like  the  other  parts  of  the  tissue,  are  made  firmer. 
In  preparing  the  isolating  solutions  it  is  better  to  dilute  the  fixing  agents  with 
normal  salt  solution  (§  331)  than  merely  with  water. 

\  260.  Isolation  by  Means  of  Formaldehyde. — Formaldehyde  in  normal 
salt  solution  is  one  of  the  very  best  dissociating  agents  for  brain  tissue  and  all  the 
forms  of  epithelium.  It  is  prepared  as  follows  :  2  cc.  of  formal,  (that  is,  a  40% 
solution  of  formaldehyde)  are  mixed  with  loco  cc.  of  normal  salt  solution.  This 
acts  quickly  and  preserves  delicate  structures  like  the  cilia  of  ordinary  epithelia, 


174 


/SOLA  TION  OF  ELEMENTS 


\CH.  VII 


and  also  of  the  endymal  cells  of  the  brain.  It  is  satisfactory  for  isolating  the 
nerve  cells  of  the  brain.  For  the  epithelium  of  the  trachea,  intestines,  etc. ,  the 
action  is  sufficient  in  two  hours  ;  good  preparations  may  also  be  obtained  after 
two  days  or  more.  The  action  on  nerve  tissue  of  the  brain  and  myel  or  spinal 
cord  is  about  as  rapid.  For  the  stratified  epithelia,  like  those  of  the  skin,  mouth, 
etc. ,  it  may  require  two  or  three  days  for  the  most  satisfactory  preparations. 


ooo 

000 
000 

ooo 
ooo 


FIG.  143  A.  FIG.  143  B.  FIG.  144. 

FIG.  143.  Preparation  Vials  for  Histology  and  Embryology.  This  repre- 
sents the  two  vials,  natural  size,  that  have  been  found  most  useful.  They  are  kept 
in  blocks  with  holes  of  the  proper  size. 

FIG.  144.     Block  with  holes  for  containing  shell  vials. 

$261.  Example  of  Isolation. — Place  a  piece  of  the  trachea  of  a  very  recently 
killed  animal,  or  the  roof  of  a  frog's  mouth,  in  the  formaldehyde  dissociator. 
After  two  hours  or  more,  up  to  two  or  three  days,  excellent  preparations  of  ciliated 
cells  may  be  obtained  by  scraping  the  trachea  or  roof  of  the  mouth  and  mounting 
the  scrapings  on  a  slide.  If  one  proceeds  after  two  hours,  probably  most  of  the 
cells  will  cling  together,  and  in  the  various  clumps  will  appear  cells  on  end  show- 
ing the  cilia  or  the  bases  of  the  cells,  and  other  clumps  will  show  the  cells  in  pro- 
file. By  tapping  the  cover  gently  with  a  needle  holder  or  other  light  object  the 
cells  will  be  more  separated  from  one  another,  and  many  fully  isolated  cells  will 
be  seen. 

\  262.  Staining  the  Cells. — Almost  any  stain  may  be  used  for  the  formalin 
dissociated  cells.  For  example,  one  may  use  eosin.  This  may  be  drawn  under 
the  cover  of  the  already  mounted  preparation  (Fig.  140),  or  a  new  preparation  may 
be  made  and  the  scrapings  mixed  with  a  drop  of  the  eosin  before  putting  on  the 
cover-glass.  It  is  an  advantage  to  study  unstained  preparations,  otherwise  one 
may  obtain  the  erroneous  opinion  that  the  structure  cannot  be  seen  unless  it  is 


CH.  VII]  ISOLATION  OF  ELEMENTS  175 

stained.     The  stain  makes  the  structural  features  somewhat  plainer  ;  it  also  accen- 
tuates some  features  and  does  not  affect  others  so  markedly. 

\  263.  Permanent  Preparations  of  Isolated  Cells. — If  one  desires  to  make  a 
permanent  preparation  of  isolated  cells  it  may  be  done  by  placing  a  drop  of  glycerin 
at  the  edge  of  the  cover  and  allowing  it  to  diffuse  under  the  cover,  or  the  diffusion 
may  be  hurried  by  using  a  piece  of  blotting  paper,  as  shown  in  Fig.  140.  One 
may  also  make  a  new  preparation  and  either  with  or  without  staining  mix  the 
cells  with  a  drop  of  glycerin  on  the  slide  and  then  cover,  or  one  may  use  glycerin 
jelly  (\  254.  326). 


FIG.  145.  Adjustable  lens  holder  with  universal  joint.  This  is  especially  use- 
ful f  o*  gross  dissections,  and  for  dissecting  the  partly  isolated  elements  with  needles 
(Leitz:  Wm.  Krafft,  N.  K). 

§  264.  Isolation  of  Musculer  Fibers. — For  this  the  formal  dissociator  may  be 
used  (\  260,  324),  but  the  nitric  acid  method  is  more  successful  (\  330).  The  fresh 
muscle  is  placed  in  this  in  a  glass  vessel.  At  the  ordinary  temperature  of  a  sitting 
room  ( 20  degrees  centigrade)  the  connective  tissue  will  be  so  far  gelatinized  in 
from  one  to  three  days  that  it  is  easy  to  separate  the  fascicles  and  fibers  either  with 
needles  or  by  shaking  in  a  test  tube  or  reagent  vial  (Fig.  143)  with  w7ater.  It 
takes  longer  for  some  muscles  to  dissociate  than  others,  even  at  the  same  temper- 
ture,  so  one  must  try  occasionally  to  see  if  the  action  is  sufficient.  When  it  is,  the 
acid  is  poured  off  and  the  muscles  washed  gently  with  water  to  remove  the  acid. 
If  one  is  ready  to  make  the  preparations  at  once  they  may  be  isolated  and  mounted 
in  water.  If  it  is  desired  to  keep  the  specimen  indefinitely  or  several  days,  the 
water  should  be  poured  off  and  a  half  saturated  solution  of  alum  added  ($  314). 
The  alum  solution  is  also  advantageous  if  the  specimens  are  to  be  stained.  The 


176  COLLODION  SECTIONING  \_CH.  VII 

specimens  may  be  mounted  in  glycerin,  glycerin  jelly  or  balsam.     Glycerin  jelly 
is  the  most  satisfactory,  however. 


FIG.  146.  Pfeiffer'1  s  preparation  microscope  with  erecting  prism  between  the 
objective  and  ocular  (Leitz  ;  Wm.  Krafft,  New  York}. 

THE  PREPARATION  OF  SECTIONS   OF  TISSUES  AND   ORGANS 

\  265.  At  the  present  time  there  are  three  principal  methods  of  obtaining 
thin  sections  of  tissues  and  organs  for  microscopic  study.  These  methods  are  : 
The  Collodion  Method,  the  Paraffin  Method,  and  the  Freezing  Method.  Each  of 
these  methods  has  its  special  application,  although  the  collodion  method  is  per- 
haps the  most  generally  applicable,  and  the  freezing  method  the  most  restricted, 
and  is  used  mostly  in  pathological  work  where  rapid  diagnosis  is  necessary  and 
the  finest  details  of  structure  are  not  so  important.  With  the  paraffin  method  the 
thinnest  sections  may  be  made,  and  in  some  ways  it  is  the  most  satisfactory  of  all. 
A  good  microtome  is  of  great  aid  in  sectioning. 

§  266.  The  Collodion  Method. — In  sectioning  by  this  method  the  tissues  are 
first  hardened  properly  and  then  entirely  infiltrated  with  collodion,  and  the  collod- 
ion hardened.  It  is  not  removed  from  the  tissue,  since  on  account  of  its  transpar- 
ency it  does  no  harm. 

\  267.  Fixing  and  Hardening  the  Tissue. — Any  of  the  approved  methods 
of  hardening  and  fixing  may  be  employed.  A  good  general  method  which  is 
applicable  to  nearly  all  of  the  tissues  and  organs  is  that  by  Picric-Alcohol.  P'or 
the  preparation  of  the  solution  see  (\  333).  A  small  piece  of  tissue  or  organ  not 


I 


CH.VI1]  COLLODION  SECTIONING  177 

containing  more  than  two  to  three  cubic  centimeters  is  placed  in  40  or  50  cc.  of 
the  picric-alcohol  and  left  6  to  24  hours,  when  the  first  picric-alcohol  should  be 
thrown  away  and  fresh  added.  After  one  or  two  days  more  the  picric- 
alcohol  should  be  poured  off  and  67%  alcohol  added.  In  a  day  or  two  this  is  re- 
placed by  75%  or  82%  alcohol  ;  82%  is  on  the  whole  most  satisfactory,  and  the 
tissue  may  be  left  in  this  till  it  is  ready  for  dehydration. 

\  268.  Dehydration  before  Infiltration. — When  one  is  ready  to  imbed  for 
sectioning,  the  tissue  must  first  be  dehydrated  in  plenty  of  95%  or  stronger  alcohol. 
It  is  better  to  take  only  a  small  piece  for  this.  The  smaller  the  piece  the  thinner 
the  sections  that  may  be  made.  The  dehydration  will  usually  be  completed  in  2  to 
24  hours.  If  the  alcohol  is  changed  two  or  three  times  the  dehydration  will  be 
hastened. 

\  269.  Saturating  with  Ether- Alcohol. (§  322). — The  next  step  is  to  remove 
the  tissue  from  the  alcohol  and  place  it  in  a  vial  of  ether-alcohol  (§  322)  for  2  to 
24  hours.  The  dehydration  becomes  somewhat  more  complete  by  this  step,  and 
the  tissue  is  more  perfectly  prepared  for  the  reception  of  the  collodion.  If  the 
dehydration  is  very  thorough  in  the  alcohol,  this  step  may  be  ommitted,  however, 
but  one  is  surer  of  success  if  the  ether-alcohol  is  used. 

\  270.  Infiltration  with  Thin  Collodion. — The  ether-alcohol  is  poured  off, 
and  a  mixture  of  thin  collodion  is  added  (2  319).  Two  or  three  hours  will  suffice 
for  objects  two  or  three  millimeters  in  thickness.  A  stay  of  one  or  more  days 
does  no  harm.  The  larger  the  object  the  more  time  is  needed. 

$  271.  Infiltration  with  Thick  Collodion. — The  thin  collodion  is  poured  off 
and  thick  collodion  (§  319)  added.  For  very  small  objects,  four  or  five  hours 
will  suffice  to  infiltrate,  but  for  larger  objects  a  longer  time  is  necessary.  The 
tissue  does  not  seem  to  be  injured  at  all  in  the  thick  collodion,  and  a  stay  in  it 
of  a  day  or  even  a  week  is  more  certain  to  insure  a  perfect  infiltration. 

\  272.  Imbedding. — The  tissue  may  be  imbedded  in  a  paper  box,  such  as  is 
used  for  paraffin  imbedding,  or  in  any  of  the  other  boxes  devised  for  paraffin.  It 
is  better,  if  paper  is  used,  to  put  a  small  amount  of  oil  on  the  paper  to  prevent 
the  collodion  from  sticking  to  it.  Vaselin  spread  over  lightly  and  then  re- 
moved, so  far  as  possible,  with  a  cloth  or  with  lens  paper,  gives  the  right  surface. 
For  small  objects  it  is  more  convenient  to  imbed  immediately  on  a  holder  that 
may  be  clamped  into  the  microtome.  Cylinders  or  blocks  of  glass,  vulcanite, 
wood  and  cork  have  all  been  recommended  and  used.  A  cork  of  the  proper  size 
is  most  convenient,  and  for  many  purposes  answers  well.  Some  collodion  is  put 
on  the  end  of  the  cork  and  a  pin  put  near  one  edge.  The  tissue  is  transferred 
from  the  thick  collodion  to  the  cork  and  leaned  against  the  pin.  Drops  of  the 
thick  collodion  are  then  poured  on  the  tissue,  and  by  moving  the  cork  properly 
the  thick  viscid  mass  may  be  made  to  surround  and  envelop  the  tissue.  Drops 
of  collodion  are  added  at  short  intervals  until  the  tissue  is  well  surrounded,  and 
then  as  soon  as  a  slight  film  hardens  on  the  surface,  the  cork  bearing  the  tissue  is 
inverted  in  a  wide-mouth  vial  of  considerably  larger  diameter  than  the  cork  (Fig. 
143).  The  vial  should  contain  sufficient  chlorform  to  float  the  cork.  The  vial  is 
then  tightly  corked.  In  imbedding  somewhat  larger  objects  on  the  end  of  a  cork 
or  other  holder,  it  is  frequently  advantageous  to  wind  oiled  paper  around  the 
holder  or  cork,  tie  it  tightly  and  have  the  projecting  hollow  cylinder  sufficiently 
long  to  receive  the  object.  The  tissue  is  then  put  into  the  cylinder  and  sufficient 


178  COLLODION  SECTIONING  [Ctf.  VII 

'collodion  added  to  completely  immerse  it.  As  soon  as  a  film  has  formed  over  the 
exposed  end,  the  cork  may  be  inverted  and  immersed  in  chloroform  as  described 
above.  For  the  use  of  "deck  plugs"  see  \  274. 

§  273.  Hardening  and  Clarifying  the  Collodion. — After  a  few  hours  the 
collodion  is  hardened  by  the  chloroform.  If  it  acts  long  enough,  and  if  no  water 
is  present,  the  imbedding  mass  is  rendered  entirely  transparent.  Whenever  the 
collodion  is  hard,  whether  it  is  clear  or  not,  the  chloroform  is  poured  off  and  the 
castor-xylene*  clarifier  (g  317)  added.  In  a  few  hours  the  imbedded  mass  will  be- 
come as  transparent  as  glass  and  the  tissue  will  seem  to  have  nothing  around  it. 
The  tissue  may  remain  for  years  in  the  castor-xylene.  Sometimes  the  collodion 
remains  white  and  opaque  for  a  considerable  time.  So  far  as  the  writer  has  been 
able  to  judge,  this  is  due  to  moisture.  If  one  breathes  on  the  mass  too  much  while 
imbedding,  or  if  it  is  very  damp  in  the  room,  opacity  may  result.  Sometimes,  in 
objects  of  considerable  size,  this  may  remain  for  a  week.  This  is  the  exception, 
however,  and  if  the  mass  seems  sufficiently  hard  and  tough,  the  cutting  may  pro- 
ceed even  if  the  clarification  is  incomplete.  | 

In  case  the  imbedding  mass  will  not  clarify  after  a  few  days  the  imbedded 
object  may  be  placed  in  95%  alcohol  for  a  day  for  dehydration,  and  then  passed 
through  chloroform  and  into  the  clarifier.  There  is  usually  no  trouble  in  getting 
the  mass  perfectly  clear  in  this  way. 

If  one  is  in  a  great  hurry,  the  collodion  may  be  hardened  in  10  or  15  minutes 
by  heating  the  bottle  containing  the  chloroform  in  a  water  bath.  The  imbedding 
block  of  hardened  collodion  may  then  be  transferred  to  the  castor-xylene  clarifier 
and  kept  warm.  It  will  soon  clear  the  collodion.  One  can  then  cut  the  sections. 

I  274.  Cutting  the  Sections.— For  cutting  the  sections  the  collodion  block  is 
usually  fastened  to  some  form  of  holder.  For  small  objects  cork  is  fairly  good. 
Blocks  of  glass,  vitrified  fiber,  etc.,  have  been  used.  If  one  uses  wood  the  "deck 
plugs"  of  the  shipwright  are  satisfactory.  They  are  about  the  right  length  when 
one  plug  is  made  into  two  holders  by  sawing  in  two  (Ewing  &  Ferguson).  To 
fasten  the  collodion  block  to  any  form  of  holder,  remove  it  from  the  castor-xylene, 
trim  as  desired,  then  dry  the  end  on  blotting  paper,  pour  some  thick  collodion  on 
the  holder  and  press  the  collodion  block  down  into  the  collodion.  The  evapora- 
tion usually  fixes  it  in  two  or  three  minutes,  when  the  holder  may  be  clamped  in 
the  jaws  of  the  microtome  and  the  cutting  proceed.  For  collodion  sectioning  a 
long,  drawing  cut  is  necessary  in  order  to  obtain  thin,  perfect  sections.  The  ob- 
ject is,  therefore,  put  in  the  jaws  of  the  microtome  at  the  right  level,  and  the 
knife  arranged  so  that  half  or  more  of  the  blade  of  the  knife  is  used  in  cutting  the 
section.  It  is  advantageous  also  to  have  the  object  with  its  long  diameter  parallel 
with  the  edge  of  the  knife.  The  surrounding  collodion  mass  should  be  cut  away, 
as  in  sharpening  a  lead  pencil,  so  that  there  is  not  more  than  a  thickness  of  about 


*The  hydrocarbon  xylene  (C8H10)  is  called  xylol  in  German.  In  English, 
members  of  the  hydrocarbon  series  have  the  termination  "ene,"  while  members 
of  the  alcohol  series  terminate  in  "ol." 

t  The  imbedded  object  may  remain  in  the  castor-xylene  clarifier  indefinitely 
without  harm.  The  collodion  grows  somewhat  tougher  by  a  prolonged  stay  in  it. 
After  cutting  all  the  sections  desired  at  one  time,  the  imbedded  tissue  is  returned 
to  the  clarifier  for  future  sections. 


CH. 


COLLODION  SECTIONING 


179 


two  millimeters  all  around  the  tissue.  This  is  to  render  the  diameter  of  the  end  to 
be  cut  as  small  as  possible.  The  smaller  the  object  the  thinner  can  the  sections  be 
made.  With  an  object  two  or  three  millimeters  thick  and  not  over  five  milli- 
meters wide,  and  a  good  sharp  knife,  sections  5^  to  6>u  can  be  cut  without  diffi- 
culty. When  knife  and  tissue  are  properly  arranged  the  tissue  and  the  knife  are 
flooded  with  the  clarifier.  Make  the  sections  with  a  steady  motion  of  the  knife. 
Then  draw  the  section  up  toward  the  back  of  the  knife  with  an  artist's  brush  and 
make  the  next  section.  Arrange  the  sections  in  serial  order  on  the  knife  blade 
till  enough  are  cut  to  fill  the  area  that  the  cover-glass  will  cover.  For  large  objects 
one  can  cut  thinner  sections  by  a  kind  of  sawing  cut. 

|  275.  Transferring  the  Sections  to  the  Slide. — If  the  clarifier  has  evaporated 
so  as  to  leave  the  sections  somewhat  dry  on  the  knife,  add  a  small  amount.  Take 
a  piece  of  thin  absorbent,  close-meshed  paper  about  twice  the  size  of  a  slide  and 
place  it  directly  upon  the  sections.  Press  the  paper  down  evenly  all  around  and 
then  pull  the  paper  off  the  edge  of  the  knife.  The  sections  will  adhere  to  the 
paper.  Place  the  paper,  sections  down,  on  a  slide,  taking  care  that  the  sections 
are  in  the  desired  position  on  the  slide.  Use  some  ordinary  lens  paper  or  some 
absorbent  paper,  and  press  it  down  gently  upon  the  transfer  paper.  This  will  ab- 
sorb the  oil,  and  then  the  transfer  paper  may  be  lifted,  with  a  rolling  motion, 
from  the  slide.  The  sections  will  remain  on  the  slide.  (See  notes  p.  180). 

§  276.  Fastening  the  Sections  to  the  Slide. — Drop  just  enough  ether- 
alcohol  (equal  parts  of  sulphuric  ether  and  95%  alchol)  on  the  sections  to  moisten 
them.  This  will  melt  the  collodion  and  fasten  the  sections  to  the  slide.  Allow  the 
slide  to  remain  in  the  air  till  the  surface  begins  to  look  slightly  dull  or  glazed. 

Sometimes,  especially  when  the  air  is  moist,  the  sections  wrinkle  badly  when 
the  ether-alcohol  is  put  on  to  fasten  them  to  the  slide.  The  excessive  wrinkling 
can  be  avoided  by  using  one  part  alcohol  and  two  parts  ether  instead  of  using 
equal  parts  of  each.  Perhaps  also  it  would  be  advantageous  in  this  case  to  use 
absolute  alcohol. 


FIG.  147.  Reagent  bottle  with  combined  cork  and  pipette  ( This 
is  made  by  taking  a  cork  of  the  proper  size  and  making  in  it  a  hole 
with  a  cork  borer  for  the  glass  tube.  It  is  advantageous  to  have  a 
string  tied  tightly  around  the  rubber  bulb  as  shown). 

\  277.  Removing  the  Oil  from  the  Sections. — As  soon  as  the 
ether-alcohol  has  evaporated  sufficiently  to  leave  the  surface  dull, 
place  the  slide  in  a  jar  of  ordinary  commercial  benzin.  It  may  be 
left  here  a  day  or  more  without  injury  to  the  sections,  but  if 
moved  around  in  the  jar  the  oil  will  be  removed  in  three  to  five 
minutes.  From  the  benzin  transfer  to  a  jar  of  95%  alcohol  to 
wash  away  the  benzin.  One  may  use  alcohol  in  the  beginning, 
but  it  dissolves  the  oil  far  less  rapidly  than  the  benzin.  The  slide 
may  remain  in  the  alcohol  half  a  day  or  more  if  one  wishes,  but  a 
stay  of  five  minutes  or  a  thorough  rinsing  of  half  a  minute  or  so 
by  moving  the  slide  around  in  the  alcohol  will  suffice. 

Xylene  is  to  be  preferred  to  benzin  for  removing  the  oil,  but 
it  is  more  expensive. 


FIG.  147. 


l8o  COLLODION  SECTIONING  \_CH.  VII 

g  278.  Staining  the  Sections  with  an  Alcoholic  Stain. — If  an  alcoholic 
stain  containing  50%  or  more  alcohol  (for  example,  hydrochloric  acid  carmine  in 
70%  alcohol)  is  used,  the  slide  may  be  removed  from  the  95%  alcohol,  drained 
somewhat  and"  then  the  stain  poured  upon  the  sections,  or  preferably,  the  slide 
immersed  in  a  jar  of  the  stain.  The  stain  is  finally  washed  away  with  67%  or 
stronger  alcohol,  the  sections  dehydrated  in  95%  alcohol,  cleared  and  mounted  in 
balsam. 

\  279.  Staining  the  Sections  with  an  Aqueous  Dye. — In  staining  with  a 
watery  stain,  the  slide  bearing  the  sections  is  transferred  from  the  95%  alcohol 
and  plunged  into  a  jar  of  water,  and  either  allowed  to  remain  a  few  minutes  or 
moved  around  in  the  water  a  moment.  Then  it  is  placed  horizontally  and  some 
of  the  stain  placed  on  the  sections  with  a  pipette,  or  preferably,  it  is  immersed  in 
a  jar  of  the  stain  ;  in  case  of  immersion  the  slide  should  stand  vertically  or  nearly 
so,  then  any  particles  of  dust,  etc. ,  in  the  stain  will  settle  to  the  bottom  of  the 
vessel  and  not  settle  on  the  sections.  When  the  sections  are  stained,  usually 
within  five  minutes,  they  are  thoroughly  washed  with  water  either  by  the  use  of  a 
pipette  or  preferably  by  immersing  in  a  jar  of  water.  They  may  then  be  counter- 
stained  for  half  a  minute  with  some  general  dye,  like  eosin  or  picric  acid,  or 
mounted  with  but  the  one  stain. t 


*Various  forms  of  paper  have  been  used  to  handle  the  collodion  sections.  It 
should 'be  moderately  strong,  fine  meshed  and  not  liable  to  shed  lint,  and  fairly 
absorbent.  One  of  the  first  and  most  successful  papers  recommended  is  "closet  or 
toilet  paper. ' '  Cigarette  paper  is  also  excellent.  In  my  own  work  the  heavy  white 
tissue  paper  has  been  found  almost  perfect  for  the  purpose.  Ordinary  lens  paper 
or  thin  blotting  paper  for  absorbing  the  oil  may  be  used  with  it.  (|  275). 

flf  one  is  a  long  time  in  cutting  a  series  of  sections,  it  sometimes  occurs  that 
the  xylene  evaporates,  and  while  the  sections  may  not  look  dry,  they  are  practically 
in  castor  oil  and  not  easily  transferable.  In  such  a  case  fresh  clarifier  or  even  a 
little  xylene  to  thin  the  oil  on  the  sections  may  be  used.  If  the  oil  is  too  thick  it 
is  viscid  and  there  is  difficulty  in  handling  the  sections  with  the  paper  as  they 
stick  rather  firmly  to  the  knife.  ($275). 

Jin  the  past  the  plan  for  changing  sections  from  95%  alcohol  to  water,  for  ex- 
ample, has  been  to  run  them  down  gradually,  using  75,  50  and  35%  alcohol,  suc- 
cessively. Each  percentage  may  vary,  but  the  principle  of  a  gradual  passing  from 
strong  alcohol  to  water  was  advocated.  On  the  other  hand  I  have  found  that  the 
safest  method  is  to  plunge  the  slide  directly  into  water  from  the  95%  alcohol.  The 
diffusion  currents  are  almost  or  quite  avoided  in  this  way.  There  is  no  time  for 
the  alcohol  and  water  to  mix,  the  alcohol  is  washed  away  almost  instantly  by  the 
flood  of  water.  So  in  dehydrating  after  the  use  of  watery  stains,  the  slide  is 
plunged  quickly  into  a  jar  of  95%  alcohol.  The  diffusion  currents  are  avoided  in 
the  same  way,  for  the  water  is  removed  by  the  flood  of  alcohol.  This  plan  has 
been  submitted  to  the  severe  test  of  laboratory  work,  and  has  proved  itself  perfectly 
satisfactory. 


CH. 


COLLODION  SECTIONING 


181 


FIG.  149. 


FIG.  148. 


FIG.  150. 


FIG.  148.  Waste  bowl  with  rack  for  supporting  slides  and  a  small  funnel  in 
which  the  slides  stand  while  draining.  This  outfit  is  easily  made  by  any  tinsmith. 
The  rack  is  composed  of  two  brass  rods  about  4  mm.  in  diameter.  The  bent  end 
pieces  are  sheet  lead.  The  funnel  is  made  of  tin,  copper  or  brass.  Either  copper 
or  brass  is  preferable  to  tin.  A  glass  dish  like  that  shown  in  Fig.  160  is  be  tier  than 
a  bowl,  as  it  can  be  more  readily  and  thoroughly  cleaned.  (Cut  loaned  by  Wm. 
Wood  &  Co. ) 


FIG.  149.     Round  glass  aquarium, 
for  all  the  uses  described  for  the  bowl. 


This  glass  vessel  is  better  than  the  bowl 
Whitall,  Tatum  &  Co.} 


FIG.  150.  Glass  box  or  ointment  jar  with  cover.  These  boxes  may  be  had  of 
various  sizes  and  can  be  used  advantageously  J  or  water,  and  for  cleaning  mixture 
for  slides  and  cover  glasses  ( \  242).  (  Whitall,  Tatum  &  Co. ) 


FIG.  151.  Section  lifter.  This  is  of  thin,  springy,  flexible  metal  placed  in  a 
handle  as  shown.  These  are  made  of  various  sizes  for  large  or  small  sections.  Such 
an  instrument  is  exceedingly  helpful  in  handling  loose  sections.  (Queen  &  Co.) 


182 


COLLODION  SECTIONING 


[CH.  VII 


FIG.  152.  Perforated  section  lifter.  This  is  easily  made  by  soldering  a  wire 
to  some  very  thin  sheet  brass  or  copper,  and  then  perforating  this  with  a  coarse 
needle  or  fine  awl.  Any  roughness  must  be  removed  by  using  a  fine  oil  stone. 

ORDER     OF     PROCEDURE     IN     MAKING     MICROSCOPICAL     PREPARATIONS     BY     THE 

COLLODION   METHOD 

\  280.  It  will  be  seen  from  this  table,  and  sections  267-281,  that  it  requires 
about  five  days  to  get  a  microscopical  preparation  if  one  commences  with  the 
fresh  tissue.  Other  methods  of  hardening  might  require  as  many  months.  It  is 
evident,  therefore,  that  one  must  exercise  foresight  in  histology  or  much  time 
will  be  wasted. 


1.  Fixing    and    hardening  the  tissue  s 

(§  267),  4  days  or  more. 

2.  Dehydrating  the  object  to  be  cut  in 
95%  or  stronger  alcohol  (\  268),  2- 
24  hours. 

3.  Saturating  the  tissue  in  ether-alcohol 
($  269),  2-24  hours. 

4.  Infiltrating  with  thin  collodion 
(\  270),  2  hours  to  2  days. 

5.  Infiltrating  in  thick  collodion($  271), 
5  hours  to  several  days. 

6.  Imbedding  the  tissue  (§272),   15  to 
20  minutes. 

7.  Hardening  the  collodion  with  chlo- 
roform (\  273),  5-24  hours. 

8.  Clarifying  and  further  hardening  the 
collodion  with  castor-xylene  ($  273), 
10-36  hours. 

9.  Cutting  the  sections  ($  274),  10  min- 
utes to  2  hours. 

10.  Transferring  the  sections  to  a  slide 
with  paper  (§  275),  i  minute. 

11.  Fastening  the  sections  to  the  slide 
with  ether-alcohol  (\  276),    i    or   2 
minutes. 


12.  Removing  the  oil  from  the  sections 
with  benzin  and  alcohol  (§  277),  3-5 
minutes,  or  24  hours. 

13.  Staining  the   sections  with  an  alco- 
holic dye   ($   278),   2  minutes  to  24 
hours.  . 

14.  Staining  the  sections  with  an  aque- 
ous dye  (g  279),  2-10  minutes. 

15.  Removing    the   superfluous   dye   by 
washing  in  water  or  alcohol  (§  278- 
279),  2-5  minutes. 

16.  Staining  with  a  general  dye  (§  279), 
15-30  seconds. 

17.  Washing   with   water   or   alcohol 
(§  278-279),  i  to  2  minutes. 

18.  Dehydrating  the  sections  in  95%  al- 
cohol (§281),  5  min.  to  24  hours. 

19.  Clearing  the  sections  (§281 ),  5  min- 
utes to  24  hours. 

20.  Draining  the  sections,   1-2  minutes. 

21.  Mounting  in  Canada  balsam  (|  281), 
1-2  minutes. 

22.  I/abeling  the  preparation  (§  308),  2 
minutes. 

23.  Cataloging  the  preparation  (\  309), 
5-10  minutes. 


CH.  VI  I  ~\ 


PARAFFIN  SECTIONING 


183 


\  281.  Mounting  in  Balsam. — After  the  sections  are  stained  they  must  be 
dehydrated  and  cleared  before  mounting  in  balsam.  For  the  dehydration  the  slide 
is  plunged  into  a  jar  of  95%  alcohol.  For  clearing  after  the  dehydration  the  slide 
is  drained  of  alcohol  and  put  down  flat  and  the  clearer  poured  on,  or  the  whole 
slide  is  immersed  in  a  jar  of  clearer  (§318).  Clearing  usually  is  sufficient  in  a 
few  minutes  ;  a  stay  of  an  hour  or  even  over  night  does  not  injure  most  sections. 

In  mounting  in  balsam  the  clearer  is  drained  away  by  standing  the  slide  nearly 
vertically  on  some  blotting  paper,  or  by  using  the  waste  bowl  and  standing  it  up 
in  the  little  funnel  (Fig.  148).  Then  the  balsam  is  put  on  the  sections  or  spread 
on  the  cover-glass  and  that  placed  over  the  sections. 

For  cataloging  and  labeling,  see  \  307-310. 


FIG.  153.  Small  spirit  lamp  modified  into  a  bal- 
sam bottle,  or  a  glycerin  or  glycerin-jelly  bottle,  or  a 
bottle  for  homogeneous  immersion  liquid.  For  all  of 
these  purposes  it  should  contain  a  glass  rod.  See  also 
Fig.  168. 


§  282.  The  Collodion  Method  with  Alcohol. — A  good  method  of  procedure 
for  making  collodion  sections  is  to  do  exactly  as  described,  including  \  272,  and 
then  instead  of  hardening  the  collodion  in  chloroform  and  clarifier,  it  is  hardened 
in  82%  alcohol  for  a  day  or  two  before  sectioning.  In  sectioning  the  knife  and 
tissue  are  kept  wet  with  82%  alcohol  and  the  sections  are  dehydrated  with  95% 
alcohol  and  then  fastened  to  the  slide  with  ether  alone  or  with  ether-alcohol. 
The  staining  and  mounting  (§  278-281)  are  as  described.  One  may  preserve  the 
tissue  after  imbedding  for  a  long  time  in  the  82%  alcohol  before  sectioning  and 
sections  may  be  made  at  any  time.  While  this  method  appears  somewhat  simpler, 
the  results  are  not  so  satisfactory  as  by  the  oil  method  given  above. 

THE    PARAFFIN    METHOD 

§  283.  As  with  the  collodion  method,  the  tissues  are  first  properly  fixed  and 
hardened  and  then  entirely  filled  with  the  imbedding  mass,  but  unlike  the  collo- 
dion the  mass  must  be  entirely  removed  before  the  sections  are  finally  mounted. 
The  tissue  thus  imbedded  and  infiltrated  is  like  a  homogeneous  mass  and  sections 
may  be  cut  of  extreme  thinness. 

§  284.  Harden  perfectly  fresh  tissue  in  picric-alcohol  (\  333)  from  one  to 
three  days.  (Any  good  method  for  fixing  and  hardening  the  elements  may  be 
used.  One  must  observe  in  each  case,  however,  the  special  conditions  necessary 


1 84 


PARAFFIN  SECTIONING 


[_CH.  VII 


for  each  method.  The  time  might  be  longer  or  shorter  than  for  the  picric-alcohol. 
(See  Lee,  the  Microtomists'  Vade-Mecum.) 

If  picric-alcohol  is  used,  pour  it  off  after  the  proper  time  for  fixing  has 
elapsed,  and  add  67%  alcohol.  Leave  this  on  the  tissue  from  one  to  three  days, 
and  if  it  becomes  very  yellow  it  is  well  to  change  it  two  or  three  times.  After  two 
or  three  days  pour  off  the  67%  alcohol  and  add  82%.  The  tissue  should  remain 
in  this  one  or  two  days,  and  it  may  remain  indefinitely. 

In  case  the  alcohol  becomes  much  yellowed,  it  should  be  changed. 

\  285.  Dehydration  and  Preparation  for  Imbedding. — From  the  pieces  of 
tissue  fixed  and  hardened  in  any  approved  manner,  cut  pieces  5  to  10  millimeters 
long  and  2  to  3  millimeters  in  breadth.  Place  one  or  two  pieces  in  a  shell  vial 
(Fig.  143.)  and  add  95%  alcohol.  Change  the  alcohol  after  two  or  three  hours, 
and  within  6  to  24  hours,  depending  on  the  size  of  the  piece  to  be  dehydrated,  the 
dehydration  will  be  completed.  The  secret  of  success  is  the  use  of  plenty  of 
alcohol  and  sufficient  time.  Absolute  alcohol  for  the  second  change  would  act 
more  promptly  and  efficiently,  but  if  plenty  of  95%  is  used  one  will  succeed, 
unless  the  day,  or  the  climate  in  general,  is  too  damp. 


FIG.  154.  Copper  pail  with 
water  bottom  for  melting  para- 
ffin. This  also  serves  as  a  water 
bath  for  large  bottles  in  which 
saturated  solutions  of  dichro- 
mate  and  other  salts  are  pre- 
pared. 


(If  one  is  studying  organs,  then  the  whole  organ  may  need  to  be  prepared  for 
imbedding,  but  for  minute  structure  small  pieces  are  preferable,  as  thinner 
sections  may  be  made. ) 

\  286.  Displacing  Alcohol  and  Clearing  Tissues  with  Cedar-wood  Oil  and 
Infiltrating  with  Paraffin. — (Lee,  p.  66.  Neelson  and  Schiefferdecker,  Arch,  fur 
Anat.  und  Physiol.,  1882,  p.  206.)  When  the  tissue  is  dehydrated  it  is  removed 
to  a  vial  of  cedar-wood  oil.  When  the  alcohol  used  for  dehydration  is  displaced 
by  the  oil,  the  tissue  will  look  clear  and  translucent.  This  requires  2  to  24  hours. 


CH.  VII} 


PARAFFIN  SECTIONING 


185 


It  is  hastened  by  warmth.  It  is  then  removed  from  the  cedar-wood  oil,  drained, 
and  placed  in  pure,  melted  paraffin,  and  this  is  then  put  into  a  paraffin  oven  and 
left  from  2  to  24  hours  (see  Ch.  X).  It  is  then  imbedded  for  sectioning.* 

Paraffin  for  infiltrating  has  usually  a  somewhat  lower  melting  point  than  that 
for  imbedding.  Equal  parts  of  paraffin  of  43  C.  and  54  C.,  answer  well.  For 
imbedding,  the  paraffin  must  be  of  a  melting  point  which  will  give  good  ribbons 
in  the  temperature  of  the  room  where  the  sectioning  is  to  be  done.  In  a  room  of 
19  to  20  C.  a  mixture  of  i  part  43  C.  paraffin  with  two  parts  of  54  C.  usually 
answers  well. 


FIG.  155.  Hot  filter  for 
paraffin,  gelatin,  balsam,  etc. 
It  is  entirely  surrounded  by  a 
•water  jacket.  The  water  is 
heated  by  placing  a  burner  under 
the  projecting  part  H.  The 
wire  basket  is  to  hold  the  strainer 
and  allow  a  free  flow  of  the 
filtered  substance  on  all  sides. 
There  is  a  bail  for  suspending 
the  filter,  and  the  filtered  sub- 
stance runs  out  through  the 
narrowed  part  F. 


\  287.  Imbedding  in  Paraffin. — Make  a  small  paper  box,  fill  it  nearly  full 
of  hot  paraffin,  place  the  box  for  a  half  minute  on  some  cold  water  to  make  a 
thin  solid  layer  on  the  bottom,  then  transfer  the  tissue  to  the  box  and  arrange 
near  one  end  so  that  the  sections  may  be  cut  in  the  desired  plane.  Now  pi  ace  the 
paper  box  on  as  cold  water  as  possible  so  that  the  paraffin  may  cool  quickly.  It 
will  be  more  homogeneous  if  cooled  quickly,  and  will  shrink  tightly  against  the 
tissue  and  avoid  air  spaces. 

In  imbedding  two  main  things  should  be  looked  after  :  The  paraffin  should 
be  hot  so  that  it  will  thoroughly  fuse  with  the  paraffin  in  the  tissue.  The  tissue 
should  be  kept  from  the  bottom  of  the  box,  either  by  holding  it  up  in  the  middle 
of  the  box  with  warmed  forceps  or  a  perforated  section  lifter  while  a  stratum 


*Thickened  cedar-wood  oil  like  that  for  oil  immersion  objectives  is  recom- 
mended by  Lee  for  clearing.  This  is  very  expensive,  and  for  most  work  unneces- 
sary. Any  form  of  cedar-wood  oil  has  been  found  satisfactory  in  the  writer's 
laboratory.  The  great  thing  is  to  have  the  tissue  thoroughly  dehydrated  before 
putting  it  into  the  oil. 


186 


PARAFFIN  SECTIONING 


[CH.  VII 


cools  on  the  bottom,  or  a  stratum  of  the  paraffin  may  be   cooled  on  the  bottom 
before  putting  the  tissue  in  the  box.     Cool  quickly  after  the  tissue  is  in  place. 


FIG.  156.  Paraffin  re- 
ceptacle P,  with  water 
bath  and  spout  for  par- 
affin imbedding  (i  88^ ) . 


\  288.  Cutting  the  Sections. — After  the  imbedding  mass  is  well  cooled,  re- 
move the  paper  box  and  trim  the  end  containing  the  tissue  in  a  pyramidal  form, 
clamp  the  block  of  paraffin  in  the  holder  of  the  microtome  so  that  the  tissue  will  be 
at  the  proper  level  for  cutting.  If  a  ribbon  microtome  is  used,  heat  the  holder  and 
melt  the  end  of  the  block  upon  it.  Cool  and  place  the  holder  in  its  place  in  the 
microtome.  Use  a  very  sharp,  dry  razor  for  cutting  the  sections.  The  sections 
are  made  with  a  rapid,  straight  cut  as  in  planing.  Do  not  try  to  section  with  a 
drawing  cut  as  in  collodion  sectioning.  If  the  temperature  of  the  room  is  right 
for  the  paraffin  used,  the  sections  will  remain  flat,  and  if  the  opposite  sides  of  the 
block  are  parallel,  and  one  edge  strikes  the  knife  squarely,  the  sections  will  adhere 
and  thus  make  a  ribbon.  If  the  room  is  too  cold  for  the  paraffin  the  sections 
will  roll.  If  it  is  too  warm  the  sections  will  crumple. 

Remember  the  sections  must  be  very  thin,  from  3//  to  is//  to  show  fine  struc- 
tural details  to  good  advantage. 

The  secret  of  making  good  ribbons  of  sections  is  to  have  the  block  of  paraffin 
containing  the  tissue  cut  square  and  properly  arranged  in  the  microtome  so  that 
the  block  strikes  the  edge  of  the  section  knife  at  right  angles  with  the  edge  ;  and 
finally  the  paraffin  must  be  of  a  proper  melting  point  for  the  room  in  which  the 
sections  are  to  be  cut.  Remember  that  the  larger  the  object  the  thicker  must 
be  the  sections,  and  the  softer  the  paraffin.  Frequently  one  may  modify  the  tem- 
perature if  too  cold  by  a  Bunsen  burner  flame  near  the  microtome.  If  it  is  too 
warm  one  may  go  to  a  basement  room. 

$  289.  Extending  Sections  with  Warm  Water. — Paraffin  sections  are  liable 
to  have  fine  wrinkles  or  folds  in  them.  These  folds  are  very  annoying  and  often 
obscure  the  structure.  To  get  rid  of  them  the  sections  are  extended  or  stretched 
upon  warm  water.  One  may  put  a  ribbon  of  sections  on  warm  water  and  then  cut 
the  ribbon  into  pieces  and  transfer  the  pieces  to  slides.  Practically,  however,  the 


CH.  F/7] 


PARAFFIN  SECTIONING 


187 


extension  is  almost  always  accomplished  on  the  slide  itself.  A  slide  is  albumen- 
ized  (|  290)  and  the  ribbon  cut  into  short  pieces  and  placed  on  the  slide.  Distilled 
or  filtered  water  is  then  added  with  a  pipette  (Fig.  147)  until  the  sections  float. 
Then  the  slide  is  moved  back  and  forth  over  an  alcohol  or  gas  flame  to  warm  the 
water.  Care  must  be  taken  to  avoid  melting  the  paraffin.  As  the  water  warms 
the  paraffin  containing  the  sections  will  flatten  and  stretch  out.  One  will  be  sur- 
prised at  the  amount  of  extension.  It  is  necessary  to  take  pieces  considerably 
shorter  than  the  cover-glass  to  be  used  or  when  extended  the  sections  will  not  all 
be  covered.  After  the  sections  are  extended,  arrange  the  ribbons  carefully  on  the 
slide  as  shown  in  Fig.  162  if  one  is  making  serial  sections.  Arrange  in  the  middle 
of  the  slide  if  only  one  or  two  sections  are  on  each  slide  (Fig.  138).  Let  the  ex- 
cess water  drain  off.  Now  let  the  slide  stand  several  hours  for  the  water  to  evapor- 
ate completely.  The  time  will  depend  on  the  temperature  and  the  dryness  of  the 
atmosphere.  If  there  is  plenty  of  time,  leave  the  slides  24  or  48  hours.  If  one 
has  a  register  with  hot  air  intake,  the  slides  may  be  put  in  the  current  of  hot  air. 
They  will  dry  out  in  half  an  hour  or  an  hour.  Sections  which  have  been  left  for  a 
year  have  given  excellent  results. 


FIG.  157.  Trays  for  slides  and  for  ribbons  of  sections.  The  figures  show  the 
construction.  It  is  important  to  have  the  bordering  f tame  with  rounded  corners  so 
that  the  trays  miy  be  easily  pulled  out  of  a  pile  or  reinserted.  The  screw  eye  shown 
in  A  makes  it  easy  to  pull  out  a  single  tray.  For  ribbons  of  sections  a  piece  of  paper 
is  placed  in  the  tray  and  the  ribbons  are  placed  on  it.  ( A )  Face  view,  ( B )  Sec- 
tional view  of  the  whole  tray,  (C)  Sectional  view  of  one  side  to  show  the  construc- 
tion more  clearly.  Trays  of  this  kind  are  so  cheap  ($15.00  pet  hundred  for  those 
holding  50  to  60  slides),  that  a  laboratory  can  have  a  great  number.  (Trans.  Amer. 
Micr.  Soc.,  1899,  p.  107). 

The  slide  trays  (Fig.  157)  are  excellent  for  drying  preparations  of  all  kinds. 
\  290.     Fastening  the  Sections  to  the  Slide. — To  fasten  the  sections  firmly 
to  the  slide,  coat  the  slide  with  albumen  fixative  (\  312)  as  follows  :     Put  a  minute 


i88 


PARAFFIN  SECTIONING 


\CH.  VII 


drop  of  the  albumen  on  the  center  of  a  slide  and  with  a  clean  finger  spread  the 
albumen  over  the  slide,  wiping  off  all  that  is  possible.  Finally  beat  or  tap  the 
slide  with  the  end  of  the  finger.  This  will  make  a  very  thin  (it  cannot  be  too 
thin)  and  even  layer. 

The  sections  are  extended  and  dried  as  described  in  $  289.  When  the  sections 
are  thoroughly  dry  they  are  in  optical  contact  with  the  slide  and  have  a  shining 
appearance  when  looking  on  the  back  of  the  slide.  When  the  sections  are  dry 
coat  them  with  $£%  collodion  made  as  follows.  Take  ^  gram  of  soluble  cotton, 
put  it  into  a  bottle  and  add  60  cc.  of  95%  or  absolute  alcohol,  and  40  cc.  of  sul- 
phuric ether.  Coat  the  sections  with  a  soft  camel's  hair  brush.  The  collodion 
should  dry  in  a  minute  or  less.  If  one  uses  too  much  ether  in  this  fixing  collo- 
dion, the  paraffin  will  be  partly  dissolved  and  will  look  moist  for  a  long  time. 
The  slight  excess  of  alcohol  will  obviate  any  such  difficulty. 


FIG.  158.  A  slide  holder  and 
bottle  for  containing  the  same  (Mix, 
Journal  of  Applied  Microscopy,  vol. 
/,  1808,  p.  160.) 


FIG.  759.  Slide  holder  with  the 
bail  hinged  so  that  it  may  be  turned 
aside  in  inserting  or  removing  the 
slides. 


When  the  collodion  is  dry  place  the  slide  in  benzin  or  xylene  to  dissolve 
the  paraffin  (see  $  291).  If  the  sections  are  not  extended  on  water,  they  may  be 
put  directly  on  the  albumenized  sides,  pressed  down  with  the  finger  and  coated 
with  collodion.  This  is  much  more  rapid,  but. does  not  get  rid  of  the  fine  folds. 
(See  Dr.  Agnes  Claypole  Trans.  Amer.  Micro.  Soc.  1894,  p.  66,  127.) 

\  291.     Removing  the  Paraffin. — Immerse  the  slide  in  a  vessel  of  xylene  or 
benzin.     This  will  dissolve  the  paraffin.     An  hour  will  usually  suffice.     One  can 


CH.  VII} 


PARAFFIN  SECTIONING 


189 


hasten  the  solution  of  the  paraffin  by  moving  the  slide  in  the  solvent.  In  this  way 
it  may  be  dissolved  in  5  to  10  minutes,  or  even  less.  It  will  do  no  harm  to  leave 
the  slide  in  the  benzin  or  xylene  over  night.  Two  or  three  days  even  might  not 
do  any  harm,  but  it  is  usually  better  to  proceed  at  once  to  the  other  operations. 


FIG.  1 60.  Apparatus  and  regents  with  which  the  slide  holders  are  used.  With 
this  apparatus  it  is  easy  to  prepare  specimens  in  large  numbers  very  expeditiously. 
After  the  sections  are  fastened  to  the  slide  and  placed  in  the  holder,  the  slides  need 
not  be  touched  during  all  the  operations  until  they  are  finally  ready  to  be  mounted 
in  balsam.  Each  holder  contains  from  12  to  14  slides.  (§277-300).  The  bottles 
for  the  reagents  are  glass  stoppered  specimen  or  museum  bottles.  (Mix,  Jour.  Ap. 
Micr.  /8o8,  p.  ///. )  Compare  Fig.  148. 

§  292.  Removing  the  Xylene  or  Benzin — From  the  xylene  or  benzin  plunge 
the  slide  bearing  the  sections  into  a  jar  of  95%  alcohol,  and  leave  it  for  a  few 
minutes,  or  move  it  around  in  the  alcohol  for  half  a  minute  or  so. 

\  293.  Staining  the  Sections  with  an  Alcoholic  Dye. — With  an  alcoholic 
stain  like  hydrochloric  acid  carmine,  remove  the  slide  from  the  alcohol,  and  add 
the  stain  directly  after  draining  the  slide,  Do  not  allow  the  stain  to  become  dry, 
for  that  would  injure  the  tissue.  Wash  away  the  stain  with  67%  alcohol,  then 
dehydrate  with  95%  alcohol,  clear  and  mount  in  balsam  as  described  below. 

\  294.  Staining  with  as  Aqueous  Dye. — Wash  away  the  95%  alcohol  from 
the  slide  bearing  the  sections  by  plunging  it  into  a  jar  of  water  and  moving  it 
around  a  moment.  Then  add  the  stain  to  the  sections  with  a  pipette,  or  immerse 
the  slide  in  a  jar  of  the  stain,  and  allow  the  stain  to  act  from  5  to  10  minutes. 
Wash  thoroughly  with  water. 

\  295.  Staining  with  a  General  Dye — Counterstaining. — If  it  is  desired  to 
give  a  general  stain  after  the  nuclear  dye  (§  294),  carmine  stained  preparations 
may  be  tinted  with  picric-alcohol  for  half  a  minute  or  more  ( \  333),  and  the  hem- 
atoxylin  stained  specimens  with  eosin  ( \  321 ).  It  usually  takes  less  than  a  minute 
for  this.  Wash  away  the  counterstain  with  water. 


190 


PARAFFIN  SECTIONING 


\_CH.  VII 


\  296.  Counterstaining  with  Picro-fuchsin.  —For  a  general  dye  to  use  with 
hematoxylin,  eosin  is  good,  but  to  differentiate  the  tissues  more  completely, 
especially  connective  tissue,  which  is  present  in  practically  every  section  made,  it 
is  better  to  use  Van  Gieson's  mixture  of  picric  acid  and  acid  fuchsin.  (Picric 
acid,  saturated  aqueous  solution  75  cc.,  water  25  cc.  i%  aqueous  solution  of  acid 
fuchsin,  10  cc.)  Sections  are  first  strongly  stained  with  hematoxylin,  well  washed 
with  water  and  then  stained  3  seconds  to  15  minutes  in  the  picro-fuchsin.  They 
are  then  washed  in  distilled  water  ;  or  in  tap  water,  to  which  has  been  added  a  drop 
or  two  of  glacial  acetic  acid  to  100  cc.  of  water.  They  are  then  dehydrated, 
cleared  and  mounted  in  acid  balsam,  that  is  in  balsam  which  has  not  been  neutral- 
ized ($  315).  If  glycerin  or  glycerin  jelly  is  used  as  amounting  medium  it  should 
be  slightly  acid.  Unless  the  mounting  medium  is  slightly  acid,  the  red  of  the 
acid  fuchsin  soon  fades.  In  some  cases  less  acid  fuchsin  should  be  used,  and  in 
some  a  greater  amount.  Acid  fuchsin  alone  without  the  picric  acid  is  also  good 
for  a  counterstain.  The  picro-fuchsin  is  a  very  valuable  differential  stain  and 
combined  in  different  proportions  with  picric  acid  will  give  great  assistance  in  al- 
most every  case.  It  does  not  seem  to  be  a  permanent  stain.  (See  Freeborn, 
Trans.  N.  Y.  Path,  Soc.,  1893,  p.  73.  Also  studies  from  the  department  of  pathol- 
ogy of  the  College  of  Physicians  and  Surgeons,  Columbia  University,  N.  Y., 
1894-1895). 

161,  Coplin's  staining  dish.  A.  The 
entire  dish ;  B.  the  dish  in  cross  section. 
This  is  made  of  glass  and  is  a  very  neat  piece 
of  apparatus.  With  it  ten  slides  may  be 
stained  at  once.  (  Whitall,  Tatum  &  Co. ) 


§  297.  Dehydration  of  the  Stained  Sec- 
tions.— Place  the  slide  with  the  stained  sec- 
tions in  a  jar  of  95%  or  absolute  alcohol  and 
leave  it  a  few  minutes,  or  wave  it  around  in 
the  alcohol  for  half  a  minute  or  so. 

Remember  that  the  larger  and  thicker 
ihe  sections  the  more  time  it  requires  to  de- 
In  a  moist  atmosphere,  95% 
alcohol  is  not  completely  satisfactory,  and 


CROSS-SECTION  hvdrate  them. 

SHOWING  SLIDES        J 


IN  POSITION. 

one  must  use  a  stronger  alcohol.     When  it  is  dry,  95%  answers  very  well. 

|  298.  Clearing  the  Sections. — Drain  off  the  alcohol,  and  place  the  slide  in  a 
jar  of  clearer  (§  318,  A  or  B)  or  put  a  drop  or  two  of  clearer  on  the  sections.  The 
clearing  is  usually  accomplished  in  two  or  three  minutes. 

§  299.  Mounting  in  Balsam. — For  this  the  clearer  is  drained  from  the 
slide,  and  wiped  away  with  blotting  paper,  cloth,  etc.  The  balsam  is  then  put 
upon  the  sections  and  the  cover  added,  or  a  cover-glass  is  spread  with  the  balsam 
and  then  put  over  the  sections.  ( If  the  sections  show  a  whitish  appearance  and 
are  opaque  they  were  not  sufficiently  dehydrated.  If  natural  balsam  is  used  the 
sections  will  clear  up  in  time). 


CH.  VII} 


SERIAL  SECTIONS 


191 


ORDER    OF     PROCEDURE     IN     MAKING     MICROSCOPICAL     PREPARATIONS     BY    THE 

PARAFFIN   METHOD 

$  300.  It  will  be  seen  from  this  table  aiid  from  sections  283  to  299,  that  it  re- 
quires from  2  to  7  days  to  get  a  microscopical  preparation  by  the  paraffin  method 
if  one  starts  with  a  fresh  tissue.  Depending  on  the  method  of  fixing  and  harden- 
ing, the  time  may  be  much  greater.  Much  time  will  be  lost  in  waiting  unless  one 
plans  ahead  in  histological  work. 


1.  Fixing  and  hardening  the  tissue  or       10. 

organ  (£  284),  4  days  or  more. 

2.  Dehydrating  the  object  to  be  cut  in   j    n. 

95%  °r  stronger  alcohol  (|  285),       12. 
i    to   24   hours    containing    %%    j 
eosin.     (§301).  13. 

3.  Displacing  the  alcohol  and  clearing 

tissues  with  cedar-wood  oil.   (See   \   14. 
§  286),  2  to  24  hours. 

4.  Infiltrating  the  tissue  with  paraffin   j    15. 

in  the  paraffin  oven  (|  286),  2  to   \ 

24  hours.  16. 

5.  Imbedding  in   paraffin  (§  287),  10 

minutes. 

6.  Cutting  the  sections  (I   288),    10   j    17. 

minutes. 

7.  Extending  the  sections  with  warm       18. 

water.     (See  I  289). 

8.  Fastening  the  sections  to  a  slide   j    19. 

(§  290),  5  minutes  to  24  hours. 

9.  Removing  the   paraffin  (§  291),  10       20. 

minutes  to  24  hours. 


Removing   the  xylene    or  benzin 

(3  292). 

Washing  with  water,  note,  p.  180. 
Staining   with     an     aqueons     dye 

(\  294),  2  minutes  to  24  hours. 
Washing     away    the     superfluous 

stain  with  water  (§  294). 
Staining  with  a  general  dye  (§  295- 

296),  10  seconds  to  10  minutes. 
Washing  the  sections  with  water 

(§  295-296). 
Dehydrating  the  stained  sections  in 

95%  alcohol  (\  297),  3  minutes  to 

24  hours. 

Clearing  the  sections  ( \  298)  2  min- 
utes to  24  hours. 
Mounting  in  Balsam  (§  299),  i  to  5 

minutes. 
Labeling  the  preparation  ( \  308) ,  2 

minutes. 
Cataloging  the  preparation  ( \  309), 

5  to  10  minutes. 


SERIAL  SECTIONS  :    IN  TOTO  STAINING. 

$301.  In  histological  studies  it  is  frequently  of  the  greatest  advantage  to 
have  the  sections  in  serial  order,  then  an  obscure  feature  in  one  section  is  fre- 
quently made  clear  by  the  following  or  preceding  sections.  While  serial  sections 
are  very  desirable  in  histological  study,  they  are  absolutely  necessary  for  the  solu- 
tion of  morphological  problems  presented  in  complex  organs  like  the  brain,  in 
embryos  and  in  minute  animals  where  gross  dissection  is  impossible. 

For  all  sections  whether  serial  or  otherwise,  if  the  sections  are  to  be  stained 
on  the  slide  it  is  of  great  advantage  to  have  the  object  stained  in  toto  in  eosin- 
during  the  dehydration  (see  2  of  the  table  above),  Very  thin  sections  and  minute 
parts  of  embryos  then  show  in  the  ribbons.  The  eosin  bulk  staining  does  not  in 
any  way  interfere  with  the  subsequent  staining  of  the  sections. 


IQ2  SERIAL  SECTIONS  [CH.  VII 

$  302.  Arrangement  of  Tissues  for  Sections  in  Histology. — The}-  should  be  so 
arranged  that  the  exact  relations  of  each  part  to  the  organ  can  be  readily  deter- 
mined. For  example,  an  organ  like  the  intestine,  a  muscle  or  a  nerve,  should  be 
so  arranged  that  exact  transections  or  longisections  can  be  made.  Organs  like  the 
liver  and  other  glands,  the  skin,  etc.,  should  be  so  arranged  that  sections  parallel 
with  the  surface  or  at  right  angles  to  it,  ( surface  or  vertical  sections)  may  be 
made.  Oblique  sections  are  often  very  puzzling. 

§  303.  Arrangement  of  Serial  Sections. — The  numerical  order  may  very  con 
veniently  be  like  the  words  on  a  printed  page,  i.  e.,  beginning  at  the  upper  left 
hand  corner  and  extending  from  left  to  right. 

The  position  of  the  various  aspects  of  the  sections  should  be  in  general  such 
that  when  they  are  under  the  compound  microscope  the  rights  and  lefts  will  corre- 
spond with  those  of  the  observer. 

\  303  a.  Imbedding  and  Sectioning  Embryos  and  Minute  Animals. — Serial 
sections  of  these  should  be  made  in  the  three  cardinal  sectional  planes,  viz  ; 
Transections  ;  Frontal  Sections  ;  Sagittal  Sections. 

If  models  are  to  be  constructed  from  the  sections  it  may  be  more  conveniently 
done  if  the  sections  are  one  of  the  following  thicknesses  :  5//.  lo/w,  i5//,  2ojii,  3O//. 
4oju,  5ow,  6o//,  8o//.  With  an  adjustable  wax-plate  tablet  any  thickness  of  sections 
may  be  modeled  ($  437). 

(A)  Transections,  that  is   sections   across  the   long  axis  of   the  embryo  or 
animal. 

Imbed  the  embryo  with  the  right  side  down,  taking  the  precautions  to  have 
a  layer  of  paraffin  between  the  embryo  and  bottom  of  the  box  (§  287). 

(1)  Mount  the  block  of  paraffin  containing  the  embryo  so  that  the  tail  end 
will  be  next  the  microtome  holder.     The  head  will  then  be  cut  first. 

(2)  Place  in  the  microtome  so  that  the  right  side  of  the  embryo  will  meet 
the  edge  of  the  knife. 

(3)  Mount  as  a  printed  line  and  the  first  or  cephalic  section  will  be  at  the 
upper  left  hand  corner,  and  the  dorsal  aspect  of  the  embryo  will  be  toward  the 
upper  edge  of  the  slide. 

Under  the  microscope  the  rights  and  lefts  will  appear  as  in  the  observer's  own 
body,  also  the  dorsal  and  ventral  aspects  so  that  he  can  easily  locate  parts  by 
comparing  them  with  his  own  body. 

(B)  Frontal  Sections,  that  is  sections  lengthwise  of  the  embryo  or  animal 
and  from  right  to  left  (dextral  and  sinistral),  so  that  the  embryo  is  divided  into 
equal  or  unequal  dorsal  and  ventral  parts. 

Imbed  the  embryo  with  the  right  side  down  in  the  imbedding  box  as  before. 

(1)  Mount  the  paraffin  block  so  that  the  ventral  side  of  the  embryo  is  next 
the  microtome  holder.     The  dorsal  side  will  then  be  cut  first. 

(2)  Let  the  right  side  of  the  embryo  meet  the  edge  of  the  knife. 

(3)  Mount  the  first  section  on  the  left  end  of  the  slide  as  before  and  so  that 
the  sections  will  be  crosswise  on  the  slide,  the  tail  toward  the  upper  edge.     Under 


CH.  VI 7 ']  SERIAL  SECTIONS  193 

the  compound  microscope  the  head  will  appear  toward  the  upper  edge  and  the 
rights  and  lefts  will  be  as  in  the  observer's  own  body. 

(4)  If  the  sections  are  too  long  to  mount  crosswise  of  the  slide,  cut  the 
sections  apart  and  mount  with  the  head  to  the  right. 

(C)  Sagittal  Sections,  that  is  sections  lengthwise  of  the  embryo  or  animal 
and  from  the  ventral  to  the  dorsal  side,   thus  dividing  the  body  into  equal  or 
unequal  right  and  left  parts. 

For  these  sections  imbed  the  embryo  with  the  right  side  down  as  before. 

1 i )  Put  the  right  side  of  the  embryo  next  the  microtome  holder,  then  the 
left  side  will  be  cut  first. 

(2)  Let  the  caudal  end  meet  the  knife  edge  if  the  embryo  is  small. 

(3)  Put  the  first  section  in  the  upper  left  hand  part  of  the  slide  as  in  the 
other  cases.     The  sections  will  be  lengthwise  of  the  slide.     This  will  bring  the 
ventral  side  up  and  the  head  to  the  right  on  the  slide.     Under  the  microscope  the 
head  will  appear  at  the  left  and  the  dorsal  side  away  from  the  observer. 

(4)  For  large  or  long  embryos  place  the  right  side  next  the  microtome  holder 
as  above,  but  let  either  dorsal  or  ventral  aspect  meet  the  knife.     Cut  the  sections 
apart  and  mount  as  in  (3). 

(D)  Axes  for  Sections. — For transections  cut  across  the  longest  straight  line 
from  head  to  tail. 

For  sagittal  sections  select  the  straightest  embryo  and  cut  parallel  with  the 
longest  axis. 

For  frontal  sections  cut  parallel  with  the  long  axis. 

$  304.  For  serial  sections  with  collodion  imbedded  objects  it  is  a  great  advan- 
tage to  have  the  imbedding  mass  unsymmetrically  trimmed,  so  that  if  a  section  is 
accidentally  turned  over  it  may  be  easily  noticed  and  rectified. 

Furthermore  it  is  imperatively  necessary  that  the  object  be  so  imbedded  that 
the  cardinal  aspects,  dextral  and  sinistral,  dorsal  and  ventral,  cephalic  and  caudal, 
shall  be  known  with  certainty. 

|  305.  Thickness  of  Cover-Glass  and  of  Serial  Sections. — It  is  a  great  advan- 
tage to  use  very  thin  cover-glasses  (0.12-0.18  mm.)  for  serial  sections,  then  the 
cover  will  not  prevent  the  use  of  high  powers.  When  the  ordinary  slides  (25  X 
76  mm.,  1X3  inch)  are  used,  cover-glasses  24  X  50  mm.  may  be  advantageously 
employed. 

The  combined  thickness  of  the  sections  on  a  slide  is  easily  determined  by  not- 
ing carefully  the  position  of  the  microtome  screw  at  the  first  and  last  sections  and 
measuring  the  elevation.  With  good  modern  automatic  microtomes  the  successive 
sections  are  almost  exactly  uniform  in  thickness,  hence  it  is  easy  to  determine  the 
combined  thickness  of  the  sections  on  a  slide  by  multiplying  the  number  of  sec- 
tions by  the  thickness  of  each. 

\  306.  Labeling  Serial  Sections. — The  label  of  a  slide  on  which  serial  sections 
are  mounted  should  contain  at  least  the  following  : 


194  SERIAL  SECTIONS  \_CN.  VII 

The  name  of  the  embryo  and  the  number  of  the  series  ;  the  number  of  the 
slide  of  that  series  ;  the  thickness  of  the  sections,  and  the  number  of  the  first  and 
last  section  on  the  slide  ;  the  date.  It  is  also  a  convenience  to  have  the  informa- 
tion repeated  in  part  on  the  left  end  and  the  number  of  the  first  section  in  each 
row,  as  shown  in  the  sample  slide  of  serial  sections.  (Fig.  211). 

LABELING,    CATALOGING   AND   STORING    MICROSCOPICAL   PREPARATIONS 

|  307.  Every  person  possessing  a  microscopical  preparation  is  interested  in 
its  proper  management  ;  but  it  is  especially  to  the  teacher  and  investigator  that 
the  labeling,  cataloging  and  storing  of  microscopical  preparations  are  of  import- 
ance. "To  the  investigator,  his  specimens  are  the  most  precious  of  his  possessions, 
for  they  contain  the  facts  which  he  tries  to  interpret,  and  they  remain  the  same 
while  his  knowledge,  and  hence  his  power  of  interpretation,  increase.  They  thus 
form  the  basis  of  further  or  more  correct  knowledge  ;  but  in  order  to  be  safe 
guides  for  the  student,  teacher,  or  investigator,  it  seems  to  the  writer  that  every 
preparation  should  possess  two  things  :  viz.,  a  label  and  a  catalog  or  history.  This 
catalog  should  indicate  all  that  is  known  of  a  specimen  at  the  time  of  its  prepara- 
tion, and  all  of  the  processes  by  which  it  is  treated.  It  is  only  by  the  possession 
of  such  a  complete  knowledge  of  the  entire  history  of  a  preparation  that  one  is 
able  to  judge  with  certainty  of  the  comparative  excellence  of  methods,  and  thus 
to  discard  or  improve  those  which  are  defective.  The  teacher,  as  well  as  the 
investigator,  should  have  this  information  in  an  accessible  form,  so  that  not  only 
he,  but  his  students  can  obtain  at  any  time,  all  necessary  information  concerning 
the  preparations  which  serve  him  as  illustrations  and  them  as  examples." 

$  308.  Labeling  Ordinary  Microscopical  Preparations. — The  label  should 
possess  at  least  the  following  information  (see  \  306  for  serial  sections): 

The  No.  of  the  preparation,  its  name  and  date  and  the  thickness  of  the  sec- 
tions and  of  the  cover-glass. 


0 

DATE. 


FIG.  163.     Example  of  a  label  of  an  ordinary  histologic 
specimen.     (See  also  Fig.  211  for  serial  sections}. 


/  /  / 

Fig.  163. 

\  309.  Cataloging  Preparations. — It  is  believed  from  personal  experience,  and 
from  the  experience  of  others,  that  each  preparation  (each  slide  or  each  series) 
should  be  accompanied  by  a  catalog  containing  at  least  the  information  suggested 
in  the  following  formula.  This  formula  is  very  flexible,  so  that  the  order  may  be 
changed,  and  numbers  not  applicable  in  a  given  case  maybe  omitted.  With  many 
objects,  especially  embryos  and  small  animals,  the  time  of  fixing  and  hardening 
may  be  months  and  even  years  earlier  than  the  time  of  imbedding.  So,  too,  an 
object  may  be  sectioned  a  long  time  after  it  was  imbedded,  and  finally  the  sections 


CH.  VII} 


LABELING  AND  CATALOGING 


195 


may  not  be  mounted  at  the  time  they  are  cut.  It  would  be  well  in  such  cases  to 
give  the  date  of  fixing  under  2,  and  under  5,  6  and  8,  the  dates  at  which  the  oper- 
ations were  performed  if  they  differ  from  the  original  date  and  from  one  another. 
In  brief,  the  more  that  is  known  about  a  preparation  the  greater  its  value. 


General  Formula  for  Cataloging  Mi- 
croscopical Preparations  : 

1.  The     general   name    and    source. 
Thickness  of  cover  glass  and  of  section. 

2.  The  number  of  the  preparation  and 
the  date  of    obtaining  and  fixing  the 
specimen  ;  the  name  of  the  preparator. 

3.  The  special  name  of  the  prepara- 
tion   and    the    common    and    scientific 
name  of  the  object  from  which  it  is  de- 
rived.    Purpose  of  the  preparation. 

4.  The  age  and  condition  of  the  ob- 
ject from  which  the  preparation  is  de- 
rived.    Condition  of    rest    or    activity ; 
fasting  or  full  fed  at  the  time  of  death. 

5.  The     chemical     treatment,  —  the 
method   of   fixing,  hardening,  dissociat- 
ing, etc.,  and  the  time  required. 

6.  The    mechanical    treatment, — im- 
bedded, sectioned,  dissected    with   nee- 
dles, etc.     Date  at  which  done. 

7.  The  staining  agent  or  agents  and 
the  time  required  for  staining. 

8.  Dehydrating   and   clearing  agent, 
mounting    medium,    cement    used     for 
sealing. 

9.  The  objectives  and  other  accesso- 
ries (micro-spectroscope,  polarizer, etc.,) 
for  studying  the  preparation. 


A  Catalog  Card  Written  According  to 
this  Formula : 

Muscular  Fibers.     Cat. 

C.  15. 
Fibers  20  to  40  n  thick. 

2.  No.  475.  (Drr.  IX)  Oct.  i,  1891.    S. 
H.  G. ,  Preparator. 

3.  Tendinous  and  intra-muscular  ter- 
minations  of     striated    muscular  fibres 
from  the  Sartorius  of  the  cat  (Felis  do- 
mestica). 

4  .  Cat  eight  months  old,  healthy  and 
well  nourished.  Fasting  and  quiet  for 
12  hours. 

5.  Muscle  pinned  on  cork  with  vas- 
elined  pin£  and  placed  in  20  per  cent, 
nitric  acid  immediately   after  death  by 
chloroform.     Left  36  hours  in  the  acid  ; 
temperature  20°   C.     In  alum  water  ()£ 
sat.  aq.  sol. )  i  day. 

6.  Fibers  separated  on  the  slide  with 
needles,  Oct.  3. 

7.  Stained  5  minutes  with  Delafield's 
hematoxylin. 

8.  Dehydrated   with   95  %   alcohol  5 
minutes,  cleared  5  minutes  with  carbol- 
turpentine,  mounted  in  xylene  balsam  ; 
sealed  with  shellac. 

9.  Use  a  1 6  mm .  for  the  general  appear- 
ance of  the  fibers,  then  a  2  or  3  mm.  ob- 
jective for  the  details  of  structure.     Try 
the  micro-polariscope  ($  218). 

10.  The  nuclei  or  muscle  corpuscles  are 
very  large  and  numerous  ;  many  of  the 
intra-muscular  ends  are  branched.  See 
S.  P.  Gage,  Proc.  Amer.  Micr.  Sci.,  1890, 
p.  132  ;  Ref.  Hand-book  Med.,  Sci.,  Vol. 
V.,p.  59- 


10.  Remarks,  including  references  to 
original  papers,  or  to  good  figures  and 
descriptions  in  books. 

\  310.  General  Remarks  on  Catalogs  and  Labels. — It  is  especially  desirable 
that  labels  and  catalogs  shall  be  written  with  some  imperishable  ink.  Some  form 
of  water-proof  carbon  ink  is  the  most  available  and  satisfactory.  The  water-proof 
India  ink,  or  the  engrossing  carbon  ink  of  Higgins,  answers  well.  As  purchased, 
the  last  is  too  thick  for  ordinary  writing  and  should  be  diluted  with  one-third  its 
volume  of  water  and  a  few  drops  of  strong  ammonia  added. 


196  LABELING  AND  CATALOGING  \_CH.  VII 

If  one  has  a  writing  diamond  it  is  a  good  plan  to  write  a  label  with  it  on  one 
end  of  the  slide.  It  is  best  to  have  the  paper  label  also,  as  it  can  be  more  easily 
read. 


FIG.  164.  Writing  diamond  for  writing  numbers  and  labels  on  glass  slides, 
cutting  cover-glasses,  etc.  ( Queen  and  Co. ) 

The  author  has  found  stiff  cards,  12^  xy^  cm.,  like  those  used  for  cataloging 
books  in  public  libraries,  the  most  desirable  form  of  catalog.  A  specimen  that  is 
for  any  cause  discarded  has  its  catalog  card  destroyed.  New  cards  may  then  be 
added  in  alphabetical  order  as  the  preparations  are  made.  In  fact  a  catalog  on 
cards  has  all  the  flexibility  and  advantages  of  the  slip  system  of  notes  (see  Wilder 
&  Gage,  p.  45). 

Some  workers  prefer  a  book  catalog.  Very  excellent  book  catalogs  have  been 
devised  by  Ailing  and  by  Ward  (Jour.  Roy.  Micr.  Soc.,  1887,  pp.  173,  348  ;  Amer. 
Monthly  Micr.  Jour.,  1890,  p.  91  ;  Amer.  Micr.  Soc.  Proc.,  1887,  p.  233). 

The  fourth  division  has  been  added  as  there  is  coming  to  be  a  strong 
belief,  practically  amounting  to  a  certainty,  that  there  is  a  different  structural 
appearance  in  many  if  not  all  of  the  tissue  elements  depending  upon  the  age  of 
the  animal,  upon  its  condition  of  rest  or  fatigue  ;  and  for  the  cells  of  the  digestive 
organs,  whether  the  animal  is  fasting  or  full  fed.  Indeed  as  physiological  histology 
is  recognized  as  the  only  true  histology,  there  will  be  an  effort  to  determine  exact 
data  concerning  the  animal  from  which  the  tissues  are  derived.  (See  Minot,  Proc. 
Amer.  Assoc.  Adv.  Science,  1890,  pp.  271-289  ;  Hodge,  on  nerve  cells  in  rest  and 
fatigue,  Jour.  Morph.,  vol.  VII.  (1892),  pp.  95-168;  Jour.  Physiol.,  vol.  XVII., 
pp.  129-134;  Gage,  The  processes  of  life  revealed  by  the  microscope  ;  a  plea  for 
physiological  histology,  Proc.  Amer.  Micr.  Soc.,  vol.  XVII.  (1895),  pp.  3-29; 
Science,  vol.  II.,  Aug.  23,  1895,  pp.  209-218. 

CABINET    FOR    MICROSCOPICAL,   PREPARATIONS 

§  311.  While  it  is  desirable  that  microscopical  preparations  should  be  pro- 
perly labeled  and  cataloged,  it  is  equally  important  that  they  should  be  protected 
from  injury.  During  the  last  few  years  several  forms  of  cabinets  or  slide  holders 
have  been  devised.  Some  are  very  cheap  and  convenient  where  one  has  but  a  few 
slides.  For  a  laboratory  or  for  a  private  collection  where  the  slides  are  numerous 
the  following  characters  seem  to  the  writer  essential  : 

( i ).  The  cabinet  should  allow  the  slides  to  lie  flat,  and  exclude  dust  and  light. 

(2).  Each  slide  or  pair  of  slides  should  be  in  a  separate  compartment.  At 
each  end  of  the  compartment  should  be  a  groove  or  bevel,  so  that  upon  depressing 
either  end  of  the  slide  the  other  may  be  easily  grasped  (Fig.  165).  It  is  also 
desirable  to  have  the  floor  of  the  compartment  grooved  so  that  the  slide  rests  only 
on  two  edges,  thus  preventing  soiling  the  slide  opposite  the  object. 

(3).  Each  compartment  or  each  space  sufficient  to  contain  one  slide  of  the 
standard  size  should  be  numbered,  preferably  at  each  end.  If  the  compartments 
are  made  of  sufficient  width  to  receive  two  slides,  then  the  double  slides  so  fre- 
quently used  in  mounting  serial  sections  may  be  put  into  the  cabinet  in  any  place 
desired. 


CH. 


LABELING  AND  CATALOGING 


197 


FIG.  165.  A  part  of  a  cabinet  drawer  seen 
from  above.  In  compartment  No.  06  is  repre- 
sented a  slide  lying  flat.  The  label  of  the 
slide  and  the  number  of  the  compartment  are 
so  placed  that  the  number  of  the  compartment 
may  be  seen  through  the  slide.  7 he  sealing 
cement  is  removed  at  one  place  to  show  that  in 
sealing  the  cover-glass^  the  cement  is  put 
partly  on  the  cover  and  partly  on  the  slide 

(I  249,  253)- 

B. — This  represents  a  section  of  the  same 
part  of  the  drawer,  (a)  Slide  resting  as  in 
a.  No.  06.  The  preparation  is  seen  to  be  above 
a  groove  in  the  floor  of  the  compartment,  (b) 
One  end  of  the  slide  is  seen  to  be  uplifted  by  de- 
pressing the  other  into  the  bevel. 

(4).  The  drawers  of  the  cabinet  should  be 
entirely  independent,  so  that  any  drawer  may 
be  partly  or  wholly  removed  without  disturb- 
ing any  of  the  others. 

(5).  On  the  front  of  each  drawer  should  be 
the  number  of  the  drawer  in  Roman  numer- 
als, and  the  number  of  the  first  and  last  com- 
partment in  the  drawer  in  Arabic  numerals 
(Fig.  166). 


FIG.  1 66.  Cabinet  for  Mi- 
croscopical Specimens,  show- 
ing the  method  of  arrange- 
ment and  of  numbering  the 
drawers  and  indicating  the 
number  of  the  first  and  last 
compartment  in  each  drawer. 
It  is  better  to  have  the  slides 
on  which  the  drawers  rest 
somewhat  shorter,  then  the 
drawer  front  may  be  entire 
and  not  notched  as  here  shown. 
(From.  Proc.  Amer.  Micr.  Soc., 
1883). 


o 


70 


FIG.  165. 


i8g  PREPARATION  OF  REAGENTS  [CH.  VII 

FIG.  167.  Slide  box  for  25  speci- 
mens. These  are  cheap  and  convenient 
and  may  be  stood  on  end  like  books, 
thus  placing  the  slides  in  a  horizontal 
position.  Smaller  boxes,  i.  e.for  j,  6 
and  12  slides  are  also  made,  and  mail- 
ing boxes  for  a  single  slide  (Bausch  & 
Lomb  Co.). 


SOME  REAGENTS  FOR   FIXING,    MOUNTING,    ETC. 

$  312.  Albumen  Fixative  (Mayer's). — This  consists  of  equal  parts  of  well- 
beaten  white  of  egg  and  glycerin.  To  each  50  cc.  of  this  I  gram  of  salicylate  of 
soda  is  added  to  prevent  putrefactive  changes.  This  must  be  carefully  filtered. 
For  method  of  use  see  $  290. 

\  313.  Alcohol  (Ethylic). — Ethyl  alcohol  is  mostly  used  for  histological  pur- 
poses. (A)  absolute  alcohol  (i.  e.,  alcohol  of  -^-§-$%}  is  recommended  for  many 
purposes,  but  if  plenty  of  95%  alcohol  is  used  it  answers  every  purpose  in 
histology,  in  a  dry  climate  or  in  a  warm,  dry  room.  When  it  is  damp  dehydration 
is  greatly  facilitated  by  the  use  of  absolute  alcohol. 

(B)  82%  alcohol  made  by  mixing  5  parts  of  95%  alcohol  with  i  part  of  water. 

(C)  67%  alcohol  made  by  mixing  2  parts  of  95%  alcohol  with  i  part  of  water. 
Grades  of  Alcohol.     It  has   been  found  by  careful  tests  that  quite  accurate 

percentages  of  alcohol  may  be  obtained  by  mixing  water  and  alcohol  as  follows  : 
Pour  alcohol  into  a  graduate  until  the  volume  of  alcohol  corresponds  to  the  de- 
sired percentage.  Add  water  until  the  volume  in  cubic  centimeters  corresponds 
to  the  original  percentage  of  the  alcohol  used.  For  example,  to  get  67%  from 
95%  alcohol,  pour  67  cc.  of  95%  into  a  graduate,  and  add  sufficient  water  to  bring 
the  volume  up  to  95  cc.  Far  50%  alcohol  from  75%,  put  50  cc.  of  75%  alcohol  in 
a  graduate,  add  sufficient  water  to  make  the  volume  75  cc.  From  the  change  in 
volume  it  does  not  answer  to  mix  given  volumes  of  water  and  alcohol  in  these 
cases.  In  the  first  case,  if  one  mixed  75  cc.  of  95%  alcohol  and  20  cc.  of  water  the 
resulting  mixture  would  be  over  75%  ;  but  if  sufficient  water  is  added  to  bring  the 
volume  back  to  the  original  percentage  more  than  20  cc.  of  water  is  added,  that  is 
enough  more  to  compenate  for  the  shrinkage,  and  the  result  is  approximately 
accurate. 

Methyl  Alcohol  is  much  cheaper  than  ethyl,  and  answers  well  in  most  micro- 
scopical processes.  It  has  recently  been  so  carefully  refined  that  the  disagreeable 
odor  is  very  little  noticeable. 

Synthol.  This  is  said  to  be  a  synthetic  form  of  alcohol.  It  seems  to  serve  the 
purpose  of  alcohol  in  histology.  Absolute  synthol  is  far  cheaper  for  the  private 
worker  than  absolute  ethyl  alcohol. 

\  314.  Alum  Solution. — For  muscle  dissociated  in  nitric  acid  use  a  satu- 
rated solution  (i.  e.,  a  solution  in  which  the  water  holds  all  the-  alum  it  can.  If 
one  adds  an  excess  so  that  there  will  always  be  some  undissolved  alum  in  the  ves- 
sel he  can  be  sure  the  solution  is  saturated  after  it  has  stood  a  few  days.  An  easy 
way  to  get  a  saturated  solution  is  to  take  500  cc.  of  water  and  add  TOO  grams  of 
alum  and  heat  the  water  in  an  agate  dish.  All  the  alum  will  be  melted,  but  on 


CH.  VII  ]  PREP  A  RA  TION  OF  RE  A  GENTS  1 99 

cooling  a  part  will  crystallize  out,  leaving  a  cold  saturated  solution).  The  satu- 
rated solution  may  be  used  but,  if  a  half  saturated  solution  is  employed,  it  will 
answer  all  the  purposes.  For  a  half  saturated  solution  take  100  cc.  of  water,  and 
100  cc.  of  saturated  alum  water  and  mix  the  two.  In  case  preparations  are  to  be 
kept  some  time  in  alum  water,  2%  of  chloral  hydrate  should  be  added  to  prevent 
mold. 

\  315.  Balsam,  Canada  Balsam,  Balsam  of  Fir;  Xvlene  Balsam. — This  is 
one  of  the  oldest  and  most  satisfactory  of  the  resinous  media  used  for  mounting 
microscopical  preparations. 

The  natural  balsam  is  most  often  used,  but  within  the  last  ten  years  the  belief 
has  arisen  that  it  is  better  to  evaporate  the  balsam  and  then  dissolve  it  in  xylene 
or  benzole.  It  certainly  dries  out  more  rapidly  if  so  treated.  Natural  balsam  has 
the  advantage  of  being  able  to  take  up  a  small  amount  of  water  so  that  if  sections 
are  not  quite  dehydrated  they  will  clear  up  after  a  time.  If  xylene  balsam  is  used 
the  dehydration  must  be  almost  complete  or  the  preparation  will  look  cloudy. 

Filtering  Balsam.  Balsam  is  now  furnished  already  filtered  through  filter 
paper.  If  one  wishes  to  filter  it  himself  a  hot  filter  like  that  shown  in  Fig.  155  is 
good.  If  xylene  balsam  is  used  it  may  be  made  thin  and  filtered  without  heat. 
For  filtering  balsam  and  all  resinous  and  gummy  materials,  the  writer  has  found  a 
paper  funnel  the  most  satisfactory.  It  can  be  used  once  and  then  thrown  away. 
Such  a  funnel  may  be  easily  made  by  rolling  a  sheet  of  thick  writing  paper 
in  the  form  of  a  cone  and  cementing  the  paper  where  it  overlaps,  or  winding  a 
string  several  times  around  the  lower  part.  Such  a  funnel  is  best  used  in  one  of 
the  rings  for  holding  funnels. 

FIG.  1 68.  Vessel  for  homogeneous  immersion  liquid 
(thick  cedar-wood  oil}.  This  is  jilled  only  a  little  above  the 
lower  end  of  the  inner  tube.  The  oil  will  not  then  run  out  if 
the  vessel  is  tipped  over.  For  applying  the  oil  there  is  a  wire 
loop  attached  to  the  upper  cork.  The  side  cork  is  for  the  pur- 
pose of  emptying  the  bottle,  and  also  for  the  escape  of  the  air 
when  filling  it.  ( The  Spencer  Lens  Co.;  see  also  Zeit. 
wiss.  Mikr. ,  /&?7,  p.  348,  and  Jour.  Roy.  Micr.  Soc. ,  1898, 
p.  238}. 


Natural  Balsam.  All  the  samples  of  balsam  tested  by  the  author  have  been 
found  slightly  acid.  This  is  an  advantage  for  carmine,  and  acid  fuchsin  stains  or 
any  other  acid  stain.  Also  for  preparations  injected  with  carmine  or  Berlin  blue. 
In  these  cases  the  color  would  fade  or  diffuse  if  the  medium  were  not  slightly 
acid.  For  hematoxylin  the  acid  is  detrimental.  For  example,  the  slight  amount 
of  acid  in  the  balsam  will  cause  the  delicate  stain  in  the  finest  fibers  of  Weigert 
preparations  to  fade.  Also  the  fuchsin  and  other  stains  which  are  faded  by  acids. 
To  neutralize  the  balsam  add  some  pure  sodium  carbonate,  set  the  balsam  in  a 
warm  place  and  shake  it  occassionally.  After  a  month  or  so  the  soda  will  settle 
and  the  clear  supernatant  balsam  will  be  found  very  slightly  alkaline.  Use  this 
whenever  an  acid  medium  would  fade  the  stain  in  the  specimen. 


200  PREPARATION  OF  REAGENTS  \CH.  VII 

\  316.  Cedar- Wood  Oil. — This  is  used  for  oil  immersion  objectives.  It  is 
best  kept  in  a  bottle  like  that  shown  in  Fig.  168.  This  oil  is  quite  thick. 

For  penetrating  tissues  and  preparing  them  for  infiltration  with  paraffin,  thick 
oil  is  recommended  by  Lee.  The  writer  has  found,  however,  that  any  good  cedar 
wood  oil  gives  excellent  results  in  ordinary  histological  and  embryological  work. 
That  known  as  Cedar  Wood  Oil  (Florida)  js  excellent,  also  that  known  as  cedar 
wood  oil  (true  Lebanon).  These  forms  are  far  less  expensive  than  that  used  for 
immersion  objectives.  The  tissues  should  be  thoroughly  dehydrated  before  put- 
ting them  into  cedar- wood  oil,  and  they  should  remain  until  they  are  transparent 
(\  286). 

\  317.  Clarifier,  Castor-Xylene  Clarifier. — This  is  composed  of  castor  oil  i 
part  and  xylene*  3  parts.  (Trans.  Amer.  Micr.  Soc.,  1895,  p.  361). 

\  318.  Clearing  Mixture  ($281,  298). — (A).  One  of  the  most  satisfactory  and 
generally  applicable  clearers  is  carbol  turpentine,  made  by  mixing  carbolic  acid 
crystals  {Acidum  carbolicum.  A.phenicum  crystallizatum]  40  cc.  with  rectified 
oil  of  turpentine  (Oleum  terebinthinae  rectificatum}  60  cc.  If  the  carbolic  acid 
does  not  dissolve  in  the  turpentine  add  5  cc.  of  95%  alcohol,  or  increase  the  tur- 
pentine, thus  :  carbolic  acid  30  cc.,  turpentine  70  cc. 

(B).  Carbol-Xylene  Clearer. — Vasale  recommends  as  a  clearer,  xylene  75  cc., 
carbolic  acid  (melted  crystals)  25  cc.  It  is  used  in  the  same  way  as  the  preceding. 

|  319.  Collodion. — This  is  a  solution  of  soluble  cottonf  or  other  form  of  pyroxy- 
lin in  equal  parts  of  sulphuric  ether  and  95%  alcohol.  Two  solutions  are  used  : 


*The  hydrocarbon  xylene  (C8H10)  is  called  xylol  in  German.  In  English 
members  of  the  hydrocarbon  series  have  the  termination  "ene"  while  members  of 
the  alcohol  series  terminate  in  "ol." 

fThe  substance  used  in  preparing  collodion  goes  by  various  names,  soluble  cot- 
ton or  collodion  cotton  is  perhaps  best.  This  is  cellulose  nitrate,  and  consists  of  a 
mixture  of  cellulose  tetranitrate  C12Hr6(NO3)4O6,  and  cellulose  pentanitrate, 
C12H15(NO3)5O5.  Besides  the  names  soluble  and  collodion  cotton,  it  is  called  gun 
cotton  and  pyroxylin.  Pyroxylin  is  the  more  general  term  and  includes  several 
of  the  cellulose  nitrates.  Celloidin  is  a  patent  preparation  of  pyroxylin,  more  ex- 
pensive than  soluble  cotton,  but  in  no  way  superior  to  it  for  imbedding. 

Soluble  cotton  should  be  kept  in  the  dark  to  avoid  decomposition.  After  it  is 
in  solution  this  decomposition  is  not  so  liable  to  occur.  The  decomposition  of  the 
dry  cotton  gives  rise  to  nitrous  acid,  and  hence  it  is  best  to  keep  it  in  a  box  loosely 
covered  so  that  the  nitrous  acid  may  escape. 

Cellulose  nitrate  is  explosive  under  concussion  and  when  heated  to  150°  centi- 
grade. In  the  air,  the  loose  soluble  cotton  burns  without  explosion.  It  is  said  not 
to  injure  the  hand  if  held  upon  it  during  ignition  and  that  it  does  not  fire  gun- . 
powder  if  burned  upon  it.  So  far  as  known  to  the  writer,  no  accident  has  ever 
occurred  from  the  use  of  soluble  cotton  for  microscopical  purposes.  I  wish  to  ex- 
press my  thanks  to  Professor  W.  R.  Orndorff,  organic  chemist  in  Cornell  Univer- 
sity, for  the  above  information.  Proc.  Amer.  Micr.  Soc.,  vol.  XVII  (1895),  pp. 
361-370. 


CH.  F/7]  PREPARATION  OF  REAGENTS  201 

(A).  6%  to  8%  or  thick  collodion.  It  is  made  by  mixing  50  cc.  of  sulphuric 
ether  and  50  cc.  of  95%  alcohol  and  adding  6  or  8  grams  of  soluble  cotton.  If  this 
is  shaken  repeatedly  the  solution  will  be  complete  in  a  day  or  two. 

(B).  1/4%  or  thin  collodion.  To  prepare  this  \y2  grams  of  soluble  cotton  are 
added  to  100  cc.  of  ether-alcohol  (§  322). 

$  320.  ^%  collodion  or  cementing  collodion.  To  prepare  it  ^ths  of  a  gram 
of  soluble  cotton  is  added  to  alcohol,  60  cc. ,  ether,  40  cc. 

The  excess  of  alcohol  prevents  the  mixture  from  softening  the  paraffin  ($  290). 

§  321.  Eosin. — This  is  used  mostly  as  a  contrast  stain  with  hematoxylin,  which 
is  an  almost  purely  nuclear  stain.  It  serves  to  stain  the  cell-body,  ground  sub- 
stance, etc.,  which  would  be  too  transparent  and  invisible  with  hematoxylin  alone. 
If  eosin  is  used  alone  it  gives  a  decided  color  to  the  tissue  and  thus  aids  in  its 
study  ($  144).  Eosin  is  used  in  alcoholic  and  in  aqueous  solutions.  A  very  satis- 
factory stain  is  made  as  follows  :  50  cc.  of  water  and  50  cc.  of  95%  alcohol  are 
mixed  and  i-ioth  of  a  gram  of  dry  eosin  added. 

The  eosin  is  used  after  the  hematoxylin  in  most  cases  (\  280),  and,  as  it  is  in 
alcoholic  solution,  it  may  be  washed  off  with  95%  alcohol  if  the  object  is  to  be 
mounted  in  balsam.  If  it  is  to  be  mounted  in  glycerin  or  glycerin  jelly,  the  excess 
of  eosin  should  be  washed  away  with  distilled  water. 

\  322.  Ether,  Ether-Alcohol. — Sulphuric  ether  is  meant  when  ether  is  men- 
tioned in  this  book.  For  the  ether-alcohol  mentioned  in  £  269,  319,  etc.,  a  mixture 
of  equal  volumes  of  sulphuric  ether  and  95%  alcohol  is  meant. 

\  323.  Farrants'  Solution. — Take  25  grams  of  clean,  dry,  gum  arabic  ;  25  cc. 
of  a  saturated  aqueous  solution  of  arsenious  acid  ;  25  cc.  of  glycerin.  The  gum 
arabic  is  soaked  for  several  days  in  the  arsenic  water,  then  the  glycerin  is  added 
and  carefully  mixed  with  the  dissolved  or  softened  gum  arabic. 

This  medium  retains  air  bubbles  with  great  tenacity.  It  is  much  easier  to 
avoid  than  to  get  rid  of  them  in  mounting. 

\  324.  Formaldehyde  Dissociator. — This  is  composed  of  2  cc.  of  a  40%  solu- 
tion of  formaldehyde  in  1000  cc.  of  water,  to  which  6  grams  of  common  table  salt 
(sodium  chlorid)  have  been  added.  Formaldehyde  as  bought  in  the  market  is  a 
40%  solution  in  water,  and  is  called  formol,  formalin,  formalose  and  formal,  the 
last  name  being  the  preferable  one.  For  its  use  in  isolating  cells  see  $  260.  (Micr. 
Bulletin  and  Sci.  News,  vol.  XII.  (1895),  pp.  4-5). 

\  325.  Glycerin. — (A).  One  should  procure  pure  glycerin  for  a  mounting 
medium.  It  needs  no  preparation,  except  in  some  cases  it  should  be  filtered 
through  filter  paper  or  absorbent  cotton  to  remove  dust,  etc. 

For  preparing  objects  for  final  mounting,  glycerin  50  cc.,  water  50  cc. ,  forms 
a  good  mixture.  For  many  purposes  the  final  mounting  in  glycerin  is  made  in  an 
acid  medium,  viz.,  Glycerin  99  cc.,  Glacial  acetic  or  formic  acid,  i  cc. 

By  extreme  care  in  mounting  and  by  occasionally  adding  a  fresh  coat  to  the 
sealing  of  the  cover-glass,  glycerin  preparations  last  a  long  time.  They  are  liable 
to  be  disappointing,  however.  In  mounting  in  glycerin  care  should  be  taken 
to  avoid  air-bubbles,  as  they  are  difficult  to  get  rid  of.  A  specimen  need  not  be 
discarded,  however,  unless  the  air-bubbles  are  large  and  numerous. 

\  326.  Glycerin  Jelly. — Soak  25  grams  of  the  best  dry  gelatin  in  cold  water  in 
a  small  agate- ware  dish.  Allow  the  water  to  remain  until  the  gelatin  is  softened. 
It  usually  takes  about  half  an  hour.  When  the  gelatin  is  softened,  as  may  be 


202  PREPARATION  OF  REAGENTS  \_CH.V1I 

readily  determined  by  taking  a  little  in  the  fingers,  pour  off  the  superfluous  water 
and  drain  well  to  get  rid  of  all  the  water  that  has  not  been  imbibed  by  the  gelatin. 
Warm  the  softened  gelatin  over  a  water  bath  and  it  will  melt  in  the  water  it  has 
absorbed.  Add  to  the  melted  gelatin  about- 5  cc.  of  egg  albumen,  white  of  egg  ; 
stir  it  well  and  then  heat  the  gelatin  in  the  water  bath  for  about  half  an  hour.  Do 
not  heat  above  75°  or  80°  C.,  for  if  the  gelatin  is  heated  too  hot  it  will  be  trans- 
formed into  meta-gelatin  and  will  not  set  when  cold.  The  heat  will  coagulate  the 
albumen  and  form  a  kind  of  floculent  precipitate  which  seems  to  gather  all  fine 
particles  of  dust,  etc.,  leaving  the  gelatin  perfectly  clear.  After  the  gelatin  is 
clarified  it  should  be  filtered  through  a  hot  flannel  filter  and  mixed  with  an  equal 
volume  of  glycerin  and  5  grams  of  chloral  hydrate  and  shaken  thoroughly.  If  it 
is  allowed  to  remain  in  a  warm  place  (i.  £,,  in  a  place  where  the  gelatin  remains 
melted)  the  air-bubbles  will  rise  and  disappear. 

In  case  the  glycerin  jelly  remains  fluid  or  semi-fluid  at  the  ordinary  temper- 
ature (i8°-2o°  C),  the  gelatin  has  either  been  transformed  into  meta-gelatin  by  too 
high  a  temperature  or  it  contains  too  much  water.  The  amount  of  water  may  be 
lessened  by  heating  at  a  moderate  temperature  over  a  waterbath  in  an  open  vessel. 
This  is  a  very  excellent  mounting  medium.  Air-bubbles  should  be  avoided  in 
mounting  as  they  do  not  disappear. 

\  327.  Chloral  Hematoxylin. — Hematoxylin  is  one  of  the  most  useful  stains 
employed  in  histology.  An  excellent  solution  for  ordinary  section  staining 
may  be  made  as  follows  :  Distilled  water  200  cc.,  and  potash  alum  i\  grams,  are 
boiled  together  for  5  minutes,  in  an  agate-ware  or  glass  vessel,  and  sufficient  boiled 
water  added  to  bring  the  water  back  to  200  cc.  After  the  mixture  is  cool,  4  grams 
of  chloral  hydrate,  and  T2oths  grams  of  hematoxylin  crystals,  previously  dissolved 
in  20  cc.  of  95%  alcohol,  are  added.  The  boiling  seems  to  destroy  any  fungi  pres- 
ent in  the  alum  or  water,  and  the  chloral  prevents  the  development  of  any  that 
may  get  in  afterward,  and  this  solution  therefore  is  quite  permanent. 

At  first  the  color  will  be  rather  faint,  but  after  a  week  or  two  it  will  become  a 
a  deep  purple.  The  deepening  of  the  color  is  more  rapid  if  the  bottle  is  left  un- 
corked in  the  light  and  is  shaken  occasionally.  It  may  be  prepared  for  work 
at  once  by  the  addition  of  a  small  amount  of  hydrogen  dioxid  (H2O2). 

If  the  stain  is  too  concentrated  it  may  be  diluted  with  freshly  distilled  water 
or  with  a  mixture  of  water,  alum  and  chloral.  If  the  stain  is  not  sufficiently  con- 
centrated, more  hematoxylin  may  be  added.  With  hematoxylin  of  the  strength 
given  in  the  formula,  sections  are  usually  sufficiently  stained  in  from  one  to  five 
minutes. 

As  may  be  inferred  from  what  was  said  above,  the  boiling  is  to  destroy  any 
living  ferments  present  in  the  water  or  alum,  and  the  chloral  hydrate  is  to  prevent 
the  development  of  germs  which  accidentally  reach  the  solution  after  it  is  made. 

No  precaution  is  necessary  in  using  this  stain  for  sections,  except  that  appli- 
cable to  all  hematoxylin  solutions,  viz  :  It  must  be  filtered  occasionally  and  after 
staining  the  surplus  stain  must  be  very  thoroughly  washed  away  with  water  ; 
otherwise  black  granules  or  needles  will  appear  in  or  upon  the  sections.  If  granules 
appear  in  the  preparations  in  spite  of  the  washing,  it  will  be  well  to  boil  the  solu- 
tion three  to  five  minutes  and  filter  through  paper  or  absorbent  cotton.  The  addi- 
tion of  one  or  two  per  cent,  of  chloral  after  the  boiling  is  also  advantageous. 
This  stain  has  not  been  tried  for  dyeing  in  bulk.  Other  substances  than  chloral 


Cff.  F/7]  PREPARATION  OF  REAGENTS  203 

were  tried,  but  not  with  so  good  success.  (Proc.  Amer.  Micr.  Soc.,  1892,  pp. 
125-127). 

\  328.  Hematein.  This  is  used  iiistead  of  hematoxylin,  as  it  is  believed  to 
give  more  satisfactory  results.  Prepare  as  follows  :  Prepare  a  5%  solution  of  pot- 
ash alum  in  distilled  water  and  boil  or  leave  in  a  steam  steralizer  an  hour  or  two. 
While  this  alum  solution  is  warm  add  i  per  cent,  of  hematein  dissolved  in  a  small 
quantity  of  alcohol.  After  the  fluid  has  cooled  add  2  grams  of  chloral  for  each 
loo  cc.  of  solution.  (Freeborn,  Jour.  Ap.  Micr.,  1900,  p.  1056. ) 

$  329.  Liquid  Gelatin. — Gelatin  or  clear  glue,  75  to  100  grams.  Commercial 
acetic  acid  (No.  8)  100  cc. ,  water  100  cc.,  or  glacial  acetic  acid  40  cc.  and  water 
160  cc.,  95%  alcohol  100  cc.,  glycerin  15  to  30  cc.  Crush  the  glue  and  put  it  into 
a  bottle  with  the  acid,  and  set  in  a  warm  place,  and  shake  occasionally.  After 
three  or  more  days  add  the  other  ingredients.  This  solution  is  excellent  for 
fastening  paper  to  glass,  wood  or  paper.  The  brush  must  be  mounted  in  a  quill 
or  wooden  handle.  For  labels,  it  is  best  to  use  linen  paper  of  moderate  thickness. 
This  should  be  coated  with  the  liquid  gelatin  and  allowed  to  dry.  The  labels  may 
be  cut  of  any  desired  size  and  attached  by  simply  moistening  them,  as  in  using 
postage  stamps. 

Very  excellent  blank  labels  are  now  furnished  by  dealers  in  microscopical 
supplies,  so  that  it  is  unnecessary  to  prepare  them  one's  self,  except  for  special 
purposes  Those  like  that  shown  in  Fig.  163  may  be  had  for  about  $4  for  10,000. 

I  330.  Nitric  Acid  Dissociator. — This  is  prepared  by  mixing  So  cc.  of  water 
with  20  cc.  of  strong  nitric  acid.  It  is  used  mostly  in  dissolving  the  connective 
tissue  of  muscle  and  thus  making  it  possible  to  separate  the  fibers.  Alum  water 
is  used  as  a  restrainer  (§314  and  264).  (Proc.  Amer.  Micr.  Soc.,  Vol.  XI, ( 1889), 
pp.  34-45). 

\  33 r-  Normal  Salt  Solution  or  Saline  Solution. — Pure  water  from  its  differ- 
ing density  from  the  natural  lymph  acts  injuriously  on  the  tissues.  The  addition 
of  a  little  table  salt,  however,  prevents  this  deleterious  action,  or  greatly  lessens  it, 
hence  the  name  of  normal  salt  solution.  It  is  a  -£$%  solution  of  table  salt  (sodium 
chlorid)  in  water  ;  water  1000  cc.,  salt  6  grams,  or  water  100  cc.,  salt  T%  gram. 

§  332.  Paraffin.  Paraffin  is  of  various  melting  points,  hence  at  the  ordinary 
temperature  of  a  laboratory,  that  melting  at  the  lowest  temperature  will  be  moder- 
ately soft,  hence  soft  paraffin,  while  that  melting  at  a  higher  temperature  will  be 
hard.  For  the  best  results  one  usually  has  to  mix  hard  and  soft  paraffins.  The 
larger  the  object  to  be  cut  and  the  thicker  the  sections  the  softer  should  be  the 
paraffin. 

\  333.  Picric-Alcohol. — This  is  an  excellent  hardener  and  fixer  for  almost  all 
tissues  and  organs.  It  is  composed  of  500  cc.  of  water  and  500  cc.  of  95%  alcohol, 
to  which  2  grams  of  picric  acid  have  been  added.  (It  is  a  i%  solution  of  picric 
acid  in  50%  alcohol).  It  acts  quickly,  in  from  one  to  three  days.  (£  267,  284). 
(Proc.  Amer.  Micr.  Soc.,  Vol.  XII  (1890),  pp.  120-122). 

\  334.  Shellac  Cement.  —Shellac  cement  for  sealing  preparations  and  for  mak- 
ing shallow  cells  (\  248)  is  prepared  by  adding  scale  or  bleached  shellac  to  95% 
alcohol.  The  bottle  should  be  filled  about  half  full  of  the  solid  shellac  then  enough 
95%  alcohol  added  to  fill  the  bottle  nearly  full.  The  bottle  is  shaken  occasionally 
and  then  allowed  to  stand  until  a  clear  stratum  of  liquid  appears  on  the  top.  This 
clear,  supernatant  liquid  is  then  filtered  through  absorbent  cotton,  using  a  paper 


204  PREPARATION  OF  REAGENTS  \_CH.VII 

funnel  (|  315),  into  an  open  dish  or  a  wide-mouth  bottle.  To  every  100  cc.  of 
this  filtered  shellac,  2  cc.  of  castor  oil  and  2  cc.  of  Venetian  turpentine  are  added 
to  render  the  shellac  less  brittle.  The  filtered  shellac  will  be  too  thin,  and  must 
be  allowed  to  evaporate  till  it  is  of  the  consistency  of  thin  syrup.  It  is  then  put 
into  a  capped  bottle,  and  for  use,  into  a  small  spirit  lamp  (Fig,  153).  In  case  the 
cement  gets  too  thick  add  a  small  amount  of  95%  alcohol  or  some  thin  shellac. 
The  solution  of  shellac  almost  always  remains  muddy,  and  in  most  cases  it  takes  a 
very  long  time  for  the  flocculent  substance  to  settle.  One  can  very  quickly  obtain  a 
clear  solution  as  follows  :  When  the  shellac  has  had  time  to  thoroughly  dissolve, 
i.  e. ,  in  a  week  or  two  in  a  warm  place,  or  in  less  time  if  the  bottle  is  frequently 
shaken,  a  part  of  the  dissolved  shellac  is  poured  into  a  bottle  and  about  one-fourth 
as  much  gasolin  or  benzin  added  and  the  two  well  shaken.  After  twenty-four 
hours  or  so  the  flocculent,  undissolved  substance  will  separate  from  the  shellac  so- 
lution and  rise  with  the  gasolin  to  the  top.  The  clear  solution  may  then  be  siphoned 
off  or  drawn  off  from  the  bottom  if  one  has  an  aspirating  bottle.  (R.  Hitchcock, 
Amer.  Monthly  Micr.  Jour  ,  July,  1884,  p.  131). 

If  one  desires  to  color  the  shellac,  the  addition  of  a  strong  alcoholic  solution 
of  some  of  the  coal  tar  colors  is  good,  but  it  is  liable  to  dissolve  in  the  mounting 
medium  when  shellac  is  used  for  sealing.     A  small  amount  of  lampblack  well 
rubbed  up  in  very  thin  shellac  and  filtered,  is  good  to  darken  the  shellac. 
1 

ARRANGING   AND    MOUNTING   MINUTE    OBJECTS 

§  335-  Minute  objects  like  diatoms  or  the  scales  of  insects  may  be  arranged  in 
geometrical  figures  or  in  some  fanciful  way.  either  for  ornament  or  more  satisfac- 
tory study.  To  do  this  the  cover-glass  is  placed  over  the  guide.  This  guide  for 
geometrical  figures  may  be  a  net-micrometer  or  a  series  of  concentric  circles.  In 
order  that  the  objects  may  remain  in  place,  however,  they  must  be  fastened  to  the 
cover-glass.  As  an  adhesive  substance,  mucilage  or  liquid  gelatin  ($  329)  thinned 
with  an  equal  volume  of  50%  acetic  acid  answers  well.  A  very  thin  coating  of  this  is 
spread  on  the  cover  with  a  needle,  or  in  some  other  wray  and  allowed  to  dry.  The  ob- 
jects are  then  placed  on  the  gelatinized  side  of  the  cover  and  carefully  got  into  posi- 
tion with  a  mechanical  finger,  made  by  fastening  a  cat's  whisker  in  a  needle  holder. 
For  most  of  these  objects  a  simple  microscope  with  stand  (Figs.  20,  145,  146)  will  be 
found  of  great  advantage.  After  the  objects  are  arranged,  one  breathes  very  gently 
on  the  cover-glass  to  soften  the  mucilage  or  gelatin.  It  is  then  allowed  to  dry  and 
if  a  suitable  amount  of  gelatin  has  been  used,  and  it  has  been  properly  moistened, 
the  objects  will  be  found  firmly  anchored.  In  mounting  one  may  use  Canada  bal- 
sam or  mount  dry  on  a  cell  (\  247,  255).  See  Newcomer,  Amer.  Micr.  Soc.'s  Proc., 
1886,  p.  128  ;  see  also  E.  H.  Griffith  and  H.  L.  Smith,  Amer.  Jour,  of  Micros.,  iv, 
102,  v,  87  ;  Amer.  Monthly  Micr.  Jour.,  i,  66.  107,  113.  Cunningham,  The  Micro- 
scope, viii,  1888,  p.  237. 


For  additional  apparatus  for  this  chapter,  see  Ch.  X. 


CHAPTER  VIII 


PHOTOGRAPHING  OBJECTS  WITH  A  VERTICAL  CAMERA  ; 
PHOTOGRAPHING  LARGE  TRANSPARENT  OBJECTS; 
PHOTOGRAPHING  WITH  A  MICROSCOPE  :  (A)  TRANS- 
PARENT OBJECTS;  (B)  OPAQUE  OBJECTS  AND  THE 
SURFACES  OF  METALS  AND  ALLOYS* 


APPARATUS   AND    MATERIAL   FOR   THIS    CHAPTER 

Vertical  camera  with  photographic  objectives  (Fig.  169),  small  vertical  camera 
with  special  microscope  stand  for  embryos,  etc.  (Figs.  183-184);  arrangement  of 
camera  for  large  transparent  objects  (Fig.  181);  photo-micrographic  cameras  ( Figs. 
183-184,  192);  photographic  objectives  for  gross  and  microscopic  work  (Figs.  170- 
171,  176-180);  microscope,  microscopic  objectives  and  projection  oculars  (Figs.  185, 
189,  193);  color  screens,  lamps,  dry  plates  and  the  chemicals  and  apparatus  neces- 
sary for  developing,  printing,  etc. 

\  336.  Nothing  would  seem  more  natural  than  that  the  camera,  armed  with  a 
photographic  objective  or  with  a  microscopic  objective,  should  be  called  into  the 
service  of  science  to  delineate  with  all  their  complexity  of  detail,  the  myriads  of 
forms  studied. 

For  photographing  many  objects  in  nature  the  camera  remains  horizontal  or 
approximately  so,  but  for  a  great  many  of  those  studied  in  botany,  zoology,  miner- 
alogy and  in  anatomy  the  specimens  cannot  be  put  in  a  vertical  position  necessary 
for  a  horizontal  camera.  This  difficulty  has  been  overcome  by  using  a  mirror  or  a 
45-degree  prism.  These  are  practically  alike  and  have  the  defect  of  producing  a 
picture  with  the  inversion  of  a  plane  mirror. 

VERTICAL   CAMERA 

$  337-  To  meet  all  the  difficulties  the  object  may  be  left  in  a  horizontal  posi- 
tion and  the  camera  made  vertical  (fig.  169). 

For  the  last  twenty-five  years  such  a  camera  has  been  in  use  in  the  Anatomical 
Department  of  Cornell  University  for  photographing  all  kinds  of  specimens  ; 


*Papers  on  this  subject  were  given  by  the  writer  at  the  meeting  of  the  Amer- 
ican Association  for  the  Advancement  of  Science  in  1879,  and  at  the  meeting  of 
the  Society  of  Naturalists  of  the  eastern  United  States  in  1883  ;  and  in  Science 
Vol.  Ill,  pp.  443,  444. 


206  PHOTO-MICROGRAPHY  [CH.  VIII 

among  these,  fresh  brains,  and  hardened  brains  have  been  photographed  without 
the  slightest  injury  to  them.  Furthermore,  as  many  specimens  are  so  delicate 
that  they  will  not  support  their  own  weight,  they  may  be  photographed  under 
alcohol  or  water  with  a  vertical  camera  and  the  result  will  be  satisfactory  as  a 
photograph  and  harmless  to  the  specimen. 

A  great  field  is  also  open  for  obtaining  life-like  portraits  of  water  animals. 
Freshly  killed  or  etherized  animals  are  put  into  a  vessel  of  water  with  a  contrast- 
ing back  ground  and  arranged  as  desired,  then  photographed.  The  fins  have 
something  of  their  natural  appearance  and  the  gills  of  branchiate  salamanders 
float  out  in  the  water  in  a  natural  way.  In  case  the  fish  tends  to  float  in  the  water 
a  little  mercury  injected  into  the  abdomen  or  intestine  will  serve  as  ballast. 

The  photographs  obtainable  in  water  are  almost  if  not  quite  as  sharp  as  those 
made  in  air.  Even  the  corrugations  on  the  scales  of  such  fishes  as  the  sucker 
(Catostomus  teres)  show  with  great  clearness.  Indeed  so  good  are  the  results 
that  excellent  half  tone  plates  may  be  produced  from  the  pictures  thus  made,  also 
excellent  photogravures.  In  those  cases,  as  in  anatomical  preparations,  where  the 
photograph  rarely  answers  the  requirements  of  a  scientific  figure,  still  a  photo- 
graph serves  as  a  most  admirable  basis  for  such  a  figure.  The  photograph  is  made 
of  the  desired  size  and  all  the  parts  are  in  correct  proportion  and  in  the  correct 
relative  position.  From  this  photographic  picture  may  be  traced  all  the  outlines 
upon  the  drawing  paper,  and  the  artist  can  devote  his  whole  time  and  energy  to 
giving  the  proper  expression  without  the  tedious  labor  of  making  measurements. 

"While  the  use  of  photography  for  outlines  as  bases  for  figures  diminishes  the 
labor  of  artists  about  one-half  it  increases  that  of  the  preparator  ;  and  herein  lies 
one  of  its  chief  merits.  The  photographs  being  exact  images  of  the  preparations, 
the  tendency  will  be  to  make  them  with  greater  care  and  delicacy,  and  the  result 
will  be  less  imagination  and  more  reality  in  published  scientific  figures  ;  and  the 
objects  prepared  with  such  care  will  be  preserved  for  future  reference." 

"In  the  use  of  photography  for  figures  several  considerations  arise  :  (i)  The 
avoidance  of  distortion  ;  (2)  The  adjustment  of  the  camera  to  obtain  an  image  of 
the  desired  size  ;  (3)  Focusing  ;  (4)  Lighting  and  centering  the  object. 

(i).  While  the  camera  delineates  rapidly,  the  image  is  liable  to.  distortion. 
I  believe  opticians  are  agreed,  that,  in  order  to  obtain  correct  photographic  images, 
the  objective  must  be  properly  made,  and  the  plane  of  the  object  must  be  parallel 
to  the  plane  of  the  ground  glass.  Furthermore,  as  most  of  the  objects  in  natural 
history  have  not  plane  surfaces,  but  are  situated  in  several  planes  at  different 
levels,  the  whole  object  may  be  made  distinct  by  using  in  the  objective  a  dia- 
phragm with  a  small  opening. 

$338.  Scale  of  Sizes  and  Focusing. — (2).  By  placing  the  camera  on  a  long 
table,  and  a  scale  of  some  kind  against  the  wall,  the  exact  position  of  the  ground 
glass  for  various  sizes  may  be  determined  once  for  all,  and  these  positions 
noted  in  some  way. 

FIG.  169.  Vertical  Camera  for  photographing  objects  in  a  horizontal  position. 
The  camera  is  attached  to  a  double  frame  connected  by  bent  metal  pieces 
fastened  to  the  lower  and  sliding  in  a  groove  in  the  upper  frame.  The  two  frames 
can  then  slide  over  each  other  without  separating.  For  moving  the  outer  frame  a 
rack  work  is  put  on  the  lower  or  inner  frame  and  a  pinion  with  a  toothed  wheel  on 
the  outer  one.  This  is  turned  by  the  wheel  shown .  To  prevent  the  camera  run- 


CH. 


PHO  TO-MICROGRAPHY 


207 


ning  down  in  the  vertical  position  a  pawl  is  held  in  place  by  a  spring.  This  may 
be  released  by  a  smaller  wheel  than  that  serving  to  move  the  pinion.  This  rack  and 
pinion  are  fine  enough  for  focusing  with  the  photographic  objectives  employed. 


FIG.  169. 

The  camera  bed  is  graduated  in  centimeters  so  that  the  exact  extent  of  the 
bellows  can  be  determined  by  inspection. 

The  support  on  which  the  specimen  rests  is  of  heavy  glass  on  vertical 
rods  about  10  centimers  long.  The  background  is  placed  on  the  table  top  about  10 
cm.  below.  This  arrangement  of  stipport  and  background  serves  to  avoid  the  dense 
shadows  which  make  it  difficult  to  determine  exactly  the  limits  of  the  specimen. 
To  make  the  apparatus  steady  the  right  hand  end  of  the  table  is  heavily  weighted. 
The  table s%  have  leveling  screws  in  the  legs. 


208 


PHO  TO-MICROGRAPHY 


\_CH.  VIII 


FIG.  170. 


FIG.  170.  Turner-Reich 
anastigmat  objective  for  pho- 
tography, (  Gundlach  Opt. 
Co.) 

In  the  camera  here  fig- 
ured, the  camera  bed  is  ruled 
in  centimeters  so  that  the 
position  of  the  ground  glass 
can  be  determined  with  ac- 
curacy and  noted.  It  takes 
but  a  moment  to  set  the 
ground  glass  or  focusing 
screen  at  the  right  level  to 
give  any  desired  size.  In 
practice  it  is  convenient  to 
have  attached  to  the  camera 
a  table  giving  the  position  of 
the  ground  glass  for  various 
sizes,  and  also  the  distance  of 
the  objective  from  the  object 
in  each  case.  By  having  this 
information  it  takes  but  a 
moment  to  set  the  camera 
and  to  place  it  so  that  it 
will  be  approximately  i  n 


FIG.  171.  Zeiss  anastigmat  objective 
for  photography ',  (Bausch  &  Lomb  Opti- 
cal Co. ) 

focus.  The  final  focusing  is  then  accom- 
plished by  the  use  of  the  rack  and  pinion 
movement.  It  is  an  advantage  to  use  a 
focusing  glass  and  a  clear  focusing  screen 
or  the  transparent  part  of  the  ordinary 
screen  (Fig.  174),  for  the  final  focusing. 
As  many  objects  have  not  sharp  details  FIG.  171. 

which  one  can  focus  on,  it  is  helpful  to 

place  some  printed  letters  on  the  part  to  be  .brought  out  with  the  greatest  sharp- 
ness.    Of  course  these  are  removed  before  the  exposure  is  made. 

§  339.  In  lighting  the  object  one  should  take  pains  to  so  arrange  it  with  ref- 
erence to  the  light  that  the  details  will  show  with  the  greatest  clearness.  Naturally 
for  the  vertical  camera  the  light  will  come  from  the  side  and  not  from  a  skylight, 
although  good  results  are  obtained  with  a  skylight  if  one  so  places  the  camera  that 
it  does  not  cast  objectionable  shadows. 


CH.  VIII} 


PHO  TO-MICROGRAPHY 


209 


FIG.  172.  Tripod  magnifier  as  focusing  glass.  This 
is  carefully  focused  on  a  scratch  or  pencil  mark  on  the 
lower  or  ground  surface  of  the  focusing  screen.  Then 
whenever  the  object  is  sharply  focused  the  focal  plane  will 
be  at  the  level  of  sensitive  surface. 

As  shown  in  Fig.  169,  the  object  is  placed  upon  a 
glass  support  and  the  background  is  quite  a  distance 
below  the  support.  For  a  dark  object  the  background 
should  be  light,  and  for  a  light  one  dark.  Black 
velveteen  is  excellent  for  a  back-ground.  The  advan-  pIG  ry2 

tage   of  the   glass  support  is  that    the  shadows  in  the 

background  which  often  make  it  difficult  to  tell  just  where  the  specimen  ends  and 
the  background  begins,  is  wholly  done  away  with,  and  that  too  without  at  all 
affecting  the  proper  light  and  shade  of  the  object  itself.  (Method  of  W.  E. 
Rumsey,  Canadian  Entomologist  1896,  p.  84). 


FIG.  173.  Focusing  Glass.  "It  is  achromatic, 
consisting  of  a  double  convex  crown  lens  and  a  nega- 
tive meniscus  flint  lens  cemented  together."  It  screws 
into  the  brass  tube  and  is  thus  adjustable,  enabling  one 
to  focus  the  pencil  mark  in  the  clear  area  of  the  focus- 
ing screen  (Fig.  174}  with  great  accuracy.  It  also 
serves  to  focus  the  image  with  ease  and  accuracy.  The 
eye  must  not  be  too  close  to  the  upper  end  ofthefocus- 
ing  glass  or  the  field  will  be  restricted.  (Gundlach 
Opt.  Co.) 


FIG.  173. 

FIG.  174.  Ground-glass  focusing  screen 
with  central  transparent  area  for  exact 
focusing  with  a  focusing  glass  when  one 
does  not  possess  a  clear  focusing  screen.  (/) 
The  ground  surface  ;  (2)  Central  part  with 
oblong  cover-glass  and  Canada  balsam  on  the 
ground  surface  to  render  it  transparent.  X. 
The  central  point  in  the  entire  focusing 
screen.  It  is  made  with  a  black  lead  pencil 
on  the  ground  surface.  The  focusing  glass 
is  focused  on  this  cross,  then  when  the  image 
is  in  focus  it  will  be  at  the  level  of  the  sen- 
sitive coating  of  the  plate . 


FIG.  175. 


CH.  VIII]  PHOTO-MICROGRAPHY  211 

FIG.  175.  Vertical  Camera  and  special  microscope  stand  for  photographing 
embryos  and  other  small  specimens  in  liquids  and  for  photographing  large  sections. 
The  camera  rests  on  a  low  table  and  the  operator  can  stand  on  the  floor  while  per- 
forming all  the  operations. 

The  stage  of  the  microscope  is  attached  to  the  arm  in  the  place  of  the  tube. 
This  stage  has  two  stories.  The  specimen  is  shown  on  the  upper  and  the  back- 
ground on  the  lower  story. 

In  focusing,  the  coarse  and  fine  adjustment  of  the  special  microscope  stand  are 
used.  The  large  mirror  is  to  illuminate  embryo  chicks  mounted  entire,  and  other 
large  transparent  preparations.  Trans.  Amer.  Micr.  1901. 

§  340.  Prints. — If  the  photographic  prints  are  to  be  used  solely  for  outlines, 
the  well-known  blue  prints  so  much  used  in  engineering  and  architecture  may  be 
made.  If,  however,  light  and  shade  and  fine  details  are  to  be  brought  out  with 
great  distinctness,  either  an  aristotype,  platinotype  or  a  bromide  print  is  preferable. 

§  341.  Recording,  Storing  and  Labeling  Negatives. — In  or- 
der to  get  the  greatest  benefit  from  past  experience  it  is  necessary  to 
make  the  results  available  by  means  of  a  careful  record.  For  this  pur- 
pose the  table  (§  360)  has  been  prepared.  If  one  gives  the  information 
called  for  in  this  table,  whether  the  result  is  successful  or  not,  one  can 
after  a  time  work  with  great  exactness,  for  the  elements  of  success  and 
failure  will  stand  out  clearly  in  the  table. 

§  342.  Labeling  the  Negatives. — After  the  negative  is  dry  the 
labeling  can  be  done  on  the  gelatin  side  with  carbon  ink.  Enough 
data  should  be  given  to  enable  the  certain  identification  of  the  negative 
at  any  future  time. 

§  343-  Storing  Negatives. — This  is  satisfactorily  done  by  put- 
ting each  into  an  envelope  and  writing  a  duplicate  label  on  the  upper 
edge,  and  then  the  negatives  may  be  placed  in  drawers  in  alphabetical 
order  as  are  the  catalog  cards  of  books  in  a  library.  One  can  then  find 
any  negative  with  the  same  facility  that  the  title  of  a  book  can  be 
found  in  a  card  catalog. 

PHOTOGRAPHING   EMBRYOS 

For  photographing  embryos  and  many  other  small  specimens  it  is 
more  convenient  to  use  a  smaller  apparatus  than  the  vertical  camera 
just  described.  It  is  necessary  also  to  have  a  more  delicate  method  of 
focusing. 

§  344.  Camera  for  Embryos. — This  is  a  vertical  camera  for 
photographing  with  the  microscope,  with  the  photographic  objective  in 
the  end  of  the  camera  as  for  an  ordinary  camera.  This  is  readily  ac- 
complished by  having  a  society  screw  adapter,  and  also  adapters  for 
the  micro-planars  or  oiher  objectives  which  one  desires  t6  use.  The 


212  PHOTO-MICROGRAPHY  \_CH.  VIII 

magnification  usually  required  varies  from  natural  size  (  X  i )  to  five 
times  natural  size  (  X  5)  up  to  X  20.  As  with  the  large  camera  the 
position  of  the  ground  glass  for  each  magnification  and  for  each  objec- 
tive is  determined  once  for  all  by  using  a  scale  in  millimeters.  The 
various  positions  are  accurately  noted,  then  one  can  set  the  camera 
almost  instantly  for  the  desired  magnification.  The  supporting  rod  is 
divided  to  half  centimeters  and  therefore  the  exact  position  is  easily 
recorded  (Fig.  183). 

FIG.  176.  Zeiss  Micro-Planar  for  photographing 
with  low  magnification  and  for  projection  (see  Ch.  IX}. 
These  are  made  from  20  to  wo  mm.  equivalent  focus, 
those  of  20  and  35  mm.  equivalent  focus  have  the  standard, 
Royal  Society  Screw,  the  others  have  a  larger  screw  in 
order  that  the  image  may  not  be  restricted.  ( Cut  loaned 
by  Bausch  &  Lomb  Optical.  Co.} 

§  345-  Special  Microscope  Stand. — For  the  accurate  focusing 
necessary  for  embryos  one  should  possess  a  special  microscope 
stand  with  the  stage  in  two  or  three  stories  and  attached  to  the  arm  in 
place  of  the  tube  of  the  microscope.  The  stage  proper  is  absent. 
This  arrangement  of  the  stage  permits  the  use  of  the  coarse  and  fine 
adjustment  of  the  microscope  to  be  used  for  focusing.  The  position  of 
the  camera  on  a  low  table  (45  to  50  cm.  high;  makes  it  possible  for  the 
operator  to  stand  on  the  floor  while  making  all  the  adjustments  of  the 
the  embryo  and  for  focusing  ;  and  all  the  parts  are  within  reach 

(Fig.  175). 

§  346.  Arranging  the  Embryos. — As  usually  prepared  the  em- 
bryos are  white  and  therefore  require  a  dark  background.  This  may 
be  attained  either  by  placing  the  embryos  in  a  dark  dish  or  on  some 
paper  blackened  with  water-proof  India  ink,  or  by  putting  them  in  a 
glass  vessel  like  a  Petri  dish,  and  a  piece  of  black  velveteen  on  the 
stage  below.  The  specimens  will  of  course  be  in  a  liquid,  usually  alcohol. 

FIG.  177.  Wide  angle  anastigmat 
rJ^__  objective  for  photographing  at  low  mag- 
'  tL  nification  (Bausch  &  Lomb  Optical  Co.}. 


If  several  embryos  are  to  be 
taken  at  once,  the  embryos  are  ar- 
ranged in  rows  something  as  the 
words  on  a  line.  Arrange  them 

in  even   vertical  as  well  as  horizontal   rows  so  that  when  the  print 
is  made  it  will  be  easy  to  cut  them  apart.     When  the  embryos  are  ar- 


CH.    VIII}  PHOTO-MICROGRAPHY  213 

ranged,  one  should  be  certain  that  the  light  brings  out  the  details  most 
desired.  For  example,  if  one  is  photographing  an  embryo  which 
shows  the  branchial  pockets  well,  great  pains  should  be  taken  to  so 
arrange  the  embryo  with  reference  to  the  light  that  the  proper  shading 
will  be  given  to  bring  out  the  gill  pockets  most  emphatically.  One  can 
learn  to  do  this  only  by  practice.  It  is  advantageous  to  have  an  assist- 
ant, then  while  the  operator  is  looking  into  the  camera  the  assistant 
can  turn  the  embryo  in  various  directions  until  the  appearance  is  most 
satisfactory. 

§  347-  Focusing  and  Making  the  Exposure. — For  getting  a 
general  focus,  and  for  the  general  arrangement  the  ground  glass  screen 
is  used,  but  for  the  final  focusing  it  is  desirable  to  use  a  clear  glass 
screen  and  a  focusing  glass.  In  this  way  one  can  focus  as  satisfactor- 
ily as  with  an  ordinary  microscope.  In  daylight  with  white  embryos 
and  a  dark  ground  30  to  40  seconds  is  usually  sufficient  exposure.  One 
must  learn  this  also  by  trial  and  it  facilitates  the  obtaining  of  exact  data 
to  make  a  record  of  every  negative  made,  whether  the  negative  is  good 
or  bad.  A  table  is  given  in  §  360  to  facilitate  the  record  taking.  In  a 
short  time  one  can  learn  to  make  the  correct  exposure.  If  the  result 
is  unsatisfactory,  try  again.  The  rule  adhered  to  by  all  first  rate 
workers  is  to  to  stick  to  it  until  the  result  is  satisfactory. 

§  348.  Records  of  Embryos. — Each  specimen  or  litter  of  speci- 
mens will  have  its  own  label  giving  date  and  method  of  preparation. 
It  is  an  advantage  to  write  this  label  with  water-proof  carbon  ink,  then 
one  can  put  the  label  in  the  dish  with  the  embryos  and  it  will  form  a 
part  of  the  picture  arid  serve  as  a  record. 

After  the  picture  is  satisfactorily  made  it  is  wise  to  number  the 
embryos  on  the  back  of  the  negative  with  a  wax  crayon,  and  later 
when  the  negative  is  dry  number  on  the  front  with  carbon  ink.  The 
embryos  are  placed  in  separate  bottles  each  with  a  copy  of  the  original 
label  and  the  number  corresponding  with  that  put  on  the  negative. 
This  is  easily  accomplished  if  the  embryos  are  arranged  in  definite 
rows  as  advised  in  §  346. 

Finally  when  the  embryo  is  cut  into  serial  sections  and  mounted, 
a  picture  of  the  whole  embryo  should  accompany  the  series. 

§  349.  Size  of  the  Pictures. — For  all  embryos  it  is  well  to 
make  one  picture  natural  size  (x  i)  and  then  for  the  smallest  ones  a 
magnification  of  at  least  five  times  natural  size  (x  5).  Here,  as  with 
the  magnification  of  the  microscope,  linear  magnification  is  always 
meant  (§  154-155). 


214 


PHO  TO-MICROGRAPH  Y 


\CH.  VIII 


§  350. — Objectives. — For  making  pictures  from  one  to  five  times 
natural  size  objectives  of  60  to  100  mm.  focus  answer  well  (Figs.  176— 
1 80).  Short  focus  (75  to  100  mm.  equivalent  focus),  wide  angle  pho- 
tographic objectives  are  also  admirable  for  this  work. 


FIG.  178.  FIG.  179. 

FIG.  178.    Leitz  64  millimeter  ob-  FIG.  179.     Leitz  42  millimeter  ob- 

jective for  photography  and  for  projec-     jective  f or  photography  and  for  projection 
tion  (  Wm.  Krafft,  N.  Y. )  (  Wm.  Krafft,  N.  Y.) 

FIG.  1 80.  Zeiss'  Apochromatic  Projection  Objective  of  70 
mm.  equivalent  focus,  for  photo-micrography.  (Zeiss'  Catalog.) 
This,  and  another  of  35  mm.  focus,  are  designed  for  making 
pictures  of  moderate  magnification.  Usually  rather  large  ob- 
jects are  photographed  with  them.  The  object  may  be  illumina- 
ted in  the  ordinary  way.  They  are  used  without  an  ocular, 
like  a  photographic  objective.  The  one  of  jj  mm.  is  screwed 
into  the  tube  of  the  microscope  like  an  ordinary  objective,  but  the 
one  of  70  mm.  here  shown,  is,  by  means  of  a  conical  adapter, 
screwed  into  the  ocular  end  of  the  tube,  Fig.  189. 

For  illuminating  the  object,  any  suitable  light  may  be  used, 
but  it  is  recommended  that  the  light  be  concentrated  by  means 
of  a  buWs  eye  or  some  form  of  combination  like  the  engraving 
glass,  and  that  the  condenser  be  so  placed  that  it  focuses  the  light  upon  the  objective, 
not  upon  the  object.    The  object  is  then  illuminated  with  a  converging  cone  of  light. 

§  351.  Record  of  Negatives. — As  indicated  in  §  341-343  each 
negative  should  have  a  record,  see  record  blank  on  p.  219.  On  the 
negative  itself  should  be  also  written  the  main  facts  with  carbon  ink. 
The  name  and  magnification,  date  and  any  other  details  which  may  be 
thought  desirable  can  be  put  on  the  envelope  containing  the  negative, 
and  then  the  negative  stored  like  a  catalog  card  as  described  above 
(§  343)- 

PHOTOGRAPHING   LARGE   TRANSPARENT    OBJECTS 

§  352.  There  are  many  large  transparent  objects  which  it  is  de- 
sirable to  photograph,  e.  g.,  chick  embryos  mounted  whole,  large  sec- 
tions of  organs  like  the  brain,  etc.  These  must  be  photographed  at  a 
low  magnification. 


CH.  VIII} 


PHO  TO-MICROGRAPH  Y 


21 


FIG.  181. 

FIG.  181.  Camera  and  special  microscope  stand  for  photographing  very  large 
transparent  sections.  For  this  the  vertical  camera  is  used  (fig.  /<5p)  with  the 
camera  reversed  on  the  sliding  frame .  This  frame  is  elevated  sufficiently  to  utilize 
the  sky  as  background  and  illuminant.  The  special  microscope  stand  is  inclined  to 
the  horizontal  and  placed  on  the  fixed  frame  supporting  the  camera  ;  the  specimen 


216  PHOTO-MICROGRAPHY  [C//.  VIII 

placed  on  the  stage.  For  objective  one  of  the  objectives  shown  in  Figs.  176  to  180  is 
used.  The  objective  is  screwed  into  an  adapter  in  place  of  the  ordinary  photographic 
objective.  The  focusing  is  performed  roughly  by  the  rack  and  pinion,  and  then 
with  great  exactness  with  the  focusing  glass.  For  manipulating  the  fine  adjust- 
ment of  the  special  microscope  the  well  known  device  of  a  cord  over  the  head  of  the 
micrometer  screw  is  used.  (See  also  Fig.  775.)  (  Trans.  Amer.  Micr.  Soc.}  igoi. ) 

Successful  photographs  require  an  even  lighting  and  an  objective 
which  has  sufficient  field  to  take  in  the  whole  object.  The  camera 
used  for  embryos  (Fig.  175)  answers  very  well  for  objects  of  moderate 
size.  For  lighting  them  the  specimen  is  put  on  the  upper  stage,  and  the 
back-ground  shown  in  the  figure  is  removed.  Then  the  large  mirror 
is  used  to  throw  light  up  through  the  preparation.  If  necessary  the 
specimen  can  be  placed  on  one  of  the  lower  stories  to  bring  it  nearer 
the  mirror.  The  lighting  and  focusing  should  be  as  perfect  as  possi- 
ble. Lamplight  and  daylight  are  both  good. 

§  353.  Photographing  Large  Transparent  Objects. — For  this 
the  large  vertical  camera  (Fig.  169,  181)  is  reversed  in  position  on  the 
supporting  frames,  and  elevated  only  sufficiently  to  make  a  sky  back- 
ground ;  or  a  45  degree  reflector  of  white  cloth  or  paper  of  sufficient 
size  must  be  used  for  a  horizontal  camera.  If  one  has  the  earth  for 
back-ground  the  light  will  be  dull  and  uneven  and  a  very  long  expos- 
ure is  necessary,  and  the  final  results  unsatisfactory. 

§  354-  Use  of  the  Special  Microscope  Stand. — In  order  to 
hold  the  specimen  in  position  and  to  focus  it  accurately,  it  is  put  on 
the  stage  of  the  special  microscope  stand  (Fig.  175),  which  is  inclined, 
and  fastened  to  the  fixed  part  of  the  frame  supporting  the  camera.  As 
the  stage  of  this  microscope  is  moved  by  the  coarse  or  the  fine  adjust- 
ment, the  focusing  can  be  accomplished  with  the  same  accuracy  as  the 
microscope  itself.  For  the  general  arrangement  of  the  specimen  and 
the  rough  focusing  the  ground  glass  is  used,  then  this  is  replaced  by  a 
clear-glass  focusing  screen,  and  by  the  aid  of  a  focusing  glass  the  speci- 
men is  put  in  perfect  focus.  As  one  cannot  reach  the  fine  adjustment 
while  focusing,  the  well  known  device  of  a  cord  over  the  head  of  the 
micrometer  screw  is  resorted  to.  The  two  ends  of  the  cord  should  be 
weighted  with  about  50  or  a  hundred  grams  to  keep  the  cord  taut,  then 
whichever  one  is  pulled,  the  micrometer  screw  will  respond  at  once. 
To  cut  off  the  light  a  piece  of  black  velveteen  is  hung  over  the  end  of 
the  objective.  This  can  be  removed  without  jarring  the  apparatus. 
An  exposure  of  a  few  seconds  (3  to  10  seconds),  will  suffice  for  many 
preparations,  unless  a  color  screen  is  used.  The  color  screen  increases 
the  time  of  exposure  from  three  to  five  times  (§  359). 


CH.  VIII} 


PHO  TO-MICROGRAPHY 


217 


FIG.  182.     BuWs  eye  lens  and  holder.     (Bausch  &  Lomb  Opt.  Co. 


COLOR-CORRECT    PHOTOGRAPHY 

From  the  fact  that  the  different  wave  lengths  of  light  affect  the  photographic 
plate  with  different  degrees  of  vigor,  the  ordinary  photographic  print  of  a  many 
colored  object  or  landscape  is  not  satisfactory.  All  objects  whose  light  is  of  short 
wave  lengths,  as  blue,  etc.,  will  appear  too  light  and  those  which  are  red,  yellow 
and  green  will  be  too  dark  relatively.  To  obviate  this  difficulty  two  methods  have 
been  adopted,  and  for  the  most  complete  success  they  must  be  combined. 

(A)  The  use  of  ortho-  or  iso- chromatic  plates  and  (B)  the  use  of  a  color  screen 
or  light  filter. 

§  355.  Orthochromatic  or  Isochromatic  Plates. — These  are  plates  which  have 
been  rendered  much  more  sensitive  than  ordinary  plates  to  the  long  waves  of  red, 
orange,  yellow  and  green,  they  therefore  give  a  much  more  natural  rendering  to 
many-colored  objects  than  ordinary  plates.  As  they  are  sensitive  to  red,  orange, 
etc. ,  one  must  be  very  careful  in  exposing  them  in  the  dark  room  even  to  the  light  of 
the  developing  lantern.  The  more  nearly  the  plate  can  be  kept  from  all  light, 
except  that  acting  during  the  exposure  in  the  camera,  the  more  satisfactory  will 
be  the  resulting  negative. 

These  color-correct  plates  are  not  very  enduring,  and  must  be  used  while  they 
are  fresh,  or  only  weak,  foggy  negatives  will  result. 


218  PHOTO-MICROGRAPHY  \_CH.  VIII 

For  photographing  transparent  or  translucent  objects  there  is  a  further  diffi- 
culty introduced,  viz,  greater  or  less  transparency.  Assuming  that  all  the  rays  of 
the  spectrum  are  equally  active,  there  would  still  be  difficulty  because  the  blue 
and  violet  stains  used  in  microscopy  are  liable  to  be  more  transparent  than  those 
stained  with  red  and  orange,  consequently  a  blue  stained  preparation  is  liable  to 
lack  in  contrast  since  the  light  reaching  the  plate  from  the  object  and  from  the 
space  around  it  produce  so  nearly  equal  effects  on  the  plate. 

On  the  other  hand  a  red  stain  gives  too  much  contrast  because  the  light  pass- 
ing through  it  has  little  effect  as  compared  with  the  light  going  through  the  space 
surrounding  the  object.  So  far  in  the  discussion  it  is  assumed  that  the  objects 
are  practically  transparent  and  that  pure  red  and  blue  are  used.  As  a  matter  of 
fact  the  stain  renders  the  specimen  somewhat  more  opaque  so  that  the  specimen 
would  be  darker  than  the  background  in  either  case.  This  is  especially  true  of 
hematoxylin.  While  hematoxylin  is  blue  or  purple,  it  renders  the  tissues  more  or 
less  opaque,  so  that  with  a  petroleum  lamp  and  isochromatic  plates,  freshly  stained 
hematoxylin  specimens  are  very  easy  to  get  good  pictures  of. 

1  356.  Color  Screens. — As  a  general  statement  all  color  screens  which  have 
proved  really  useful  in  photographing  transparent  or  translucent  microscopic 
specimens  cut  off  most  of  the  blue  end  of1  the  spectrum.  Others  cut  off  also  the 
red  end,  leaving  only  the  middle,  visually  brightest  part  of  the  spectrum  free.  As 
modern  achromatic  objectives  are  corrected  for  the  visual  rays,  a  screen  cutting 
off  the  blue  end  of  the  spectrum  serves  to  obviate  any  lack  of  sharpness  due  to 
the  aberration  of  the  blue  rays  in  such  objectives.  There  is  the  further  advan- 
tage that  with  red  and  yellow  objects,  the  color  being  in  general  of  the  wave 
length  transmitted  by  the  screen  would  be  in  true  relative  shade  or  contrast  be- 
tween background  and  object,  and  give  good  detail  in  the  object.  For  the  blue 
object  this  form  of  screen  is  also  good  for,  as  it  cuts  off  the  blue  rays,  the  effect 
will  be  like  photographing  a  gray  object  where  the  light  and  shade  depend  on  the 
transparency  of  different  parts.  Thfc  denser  the  part  the  more  opaque  it  is  and 
therefore  the  darker  it  appears  with  transmitted  light.  The  background  allowing 
all  the  colored  light  to  pass  is  lighter  than  the  blue  object  and  therefore  there  will 
be  good  contrast  and  also  good  detail  in  the  object. 

\  357  Composition  of  Color  Screens. — The  most  successful  color  screens  are 
solutions  held  in  parallel  sided  glass  vessels.  Colored  glass,  gelatin,  collodion, 
etc.,  colored  with  different  chemicals  are  fairly  satisfactory,  but  not  so  satisfac- 
tory as  the  solutions. 

(1)  The  most  generally  used  and  most  generally  useful  screen  is  a  watery 
solution  of  dichromate  of  potash  (K2Cr2O7).     This  cuts  off  the  violet,  the  blue 
and  the  bluish  green.  The  amount  of  light  cut  out  depends  upon  the  density  of  the 
solution  and  the  thickness  of  the  stratum  through  which  the  light  passes. 

(2)  Zettnow' s  cupro-chr ornate  color  screen  allows  wave  lengths  from  0.570^ 
to  0.550/1  to  pass,  that  is  yellow  light.     It  is  made  by  dissolving  in  250  cc.  of  water, 
ii  grams  of  pure  dry  cupric  nitrate  and  i  gram  of  chromic  acid.     The  thickness  of 
the  stratum  of  liquid  should  be  about  i  centimeter. 

(3)  Gifford's  color  screen.     This  is  highly  spoken  of  both  for  observation 
with  the  microscope  (Carpenter-Dallinger)  and  for  use  in  photography  (J.  R.  M. 
S.  1894,  p.  164).     It  is  composed  of  a  strong  solution  of  malachit  green  in  water, 
glycerin,  glycerin  jelly,  etc.     Only  a  thin  stratum  such  as  could  be  mounted  be- 


HI 


o 
•     «! 


a  1 1 


_    X      . 

f  I 


511 


5-0  0 


M 


220  PHOTO-MICROGRAPHY  \CH.  VIII 

tween  two  cover-glasses  is  needed.  By  combining  a  little  picric  acid  with  the 
solution  or  by  the  use  of  a  thin  piece  of  signal  green  glass  only  light  between  the 
fixed  lines  E  and  F,  is  allowed  to  pass.  This  is  not  therefore  so  generally  useful 
as  dichromate. 

(4)  Bothamley's  aurantia  color  screen  is  a  saturated  alcoholic  solution  of  the 
aurantia  added  to  collodion  of  3  to  4%.  The  collodion  is  poured  on  a  large  cover- 
glass  or  a  glass  plate  and  allowed  to  dry.  Pringle  advises  several  screens  of  aurantia 
of  different  shades.  That  is  easily  managed  by  adding  a  greater  or  less  amount  of 
the  solution  to  the  collodion.  This  is  a  good  screen  and  easily  used. 

Petroleum  light  serves  as  a  yellow  color  screen,  and  one  can  often  get  excel- 
lent results  with  such  a  light  when  daylight  or  the  electric  light  without  a  color 
screen  does  not  give  a  good  picture.  For  all  photography  with  the  microscope 
isochromatic  or  orthochromatic  plates  are  advised.  For  many  objects  no  color 
screen  is  needed  if  one  uses  a  petroleum  lamp. 

$  358.  Position  of  the  Screen. — It  does  not  make  much  difference  where  the 
color  screen  is  placed  provided  no  light  reaches  the  object  which  has  not  passed 
through  the  screen. 

\  359.  Exposure  with  a  Color  Screen. — The  interposition  of  a  color  screen 
increases  the  time  of  exposure  from  three  to  five  times.  One  can  learn  the  time 
and  whether  or  not  to  use  a  color  screen,  and  the  kind  of  a  screen  to  use  only  by 
experiment.  To  get  the  full  benefit  of  these  experiments  for  future  work,  every 
negative  should  be  carefully  recorded  (\  360,  table).  It  would  also  aid  one  materi- 
ally, in  the  beginning  at  least,  if  he  were  to  study  the  color  screen  used  with  the 
micro-spectroscope  and  determine  the  wave  lengths  which  are  allowed  to  pass 
through  it  (\  195,  202).  If  this  study  were  supplemented  by  a  spectroscopic  ex- 
amination of  the  object  to  be  photographed,  one  would  learn  to  choose  with  great 
accuracy  the  color  screen  which  would  give  the  best  results. 

PHOTOGRAPHING   WITH    A    MICROSCOPE* 

$  361.  The  first  pictures  jnade  on  white  paper  and  white  leather,  sensitized 
by  silver  nitrate,  were  made  by  the  aid  of  a  solar  microscope  ( 1802).  The  pictures 


^Considerable  confusion  exists  as  to  the  proper  nomenclature  of  photography 
with  the  microscope.  In  German  and  French  the  term  micro-photography  is  very 
common,  while  in  English  photo-micrography  and  micro-photography  mean  dif- 
ferent things.  Thus  :  A  photo-micrograph  is  a  photograph  of  a  small  or  microscopic 
object  usually  made  with  a  microscope  and  of  sufficient  size  for  observation  with 
the  unaided  eye  ;  while  a  micro-photograph  is  a  small  or  microscopic  photograph 
of  an  object,  usually  a  large  object,  like  a  man  or  woman  and  is  designed  to  be 
looked  at  with  a  microscope. 

Dr.  A.  C.  Mercer,  in  an  article  in  the  Proc.  Amer.  Micr.  Soc.,  1886,  p.  131,  says 
that  Mr.  George  Shadbolt  made  this  distinction..  See  the  Liverpool  and  Manches- 
ter Photographic  Journal  (now  British  Journal  of Photography} ,  Aug.  15,  1858,  p. 
203  ;  also  Button's  Photographic  Notes,  Vol.  Ill,  1858,  pp.  205-208.  On  p.  208  of 
the  last,  Shadbolt's  word  "Photomicrography"  appears.  Dr.  Mercer  puts  the 
case  very  neatly  as  follows  :  "A  photo-micrograph  is  a  macroscopic  photograph  of 
a  microscopic  object  ;  a  micro-photograph  is  a  microscopic  photograph  of  a  macro- 
scopic object.  See  also  Medical  News,  Jan.  27,  1894,  p.  108. 


CH.  VIII}  PHOTO-MICROGRAPHY  221 

were  made  by  Wedgewood  and  Davy,  and  Davy  says  :  "I  have  found  that  images 
of  small  objects  produced  by  means  of  the  solar  microscope  may  be  copied  without 
difficulty  on  prepared  paper,  "t 

Thus  among  the  very  first  of  the  experiments  in  photography  the  microscope 
was  called  into  requisition.  And  naturally,  plants  and  motionless  objects  were 
photographed  in  the  beginnings  of  the  art  when  the  time  of  exposure  required 
was  very  great. 

At  the  present  time  photography  is  used  to  an  almost  inconceivable  degree  in 
all  the  arts  and  sciences  and  in  pure  art.  Even  astronomy  finds  it  of  the  greatest 
assistance. 

It  has  also  accomplished  marvels  in  the  production  of  colored  plates  for  book 
illustrations,  especially  in  natural  history.  For  an  example  see  Comstock's  Insect 
Life,  2d  edition. 

Although  first  in  the  field,  Photo-Micrography  has  been  least  successful  of 
the  branches  of  photography.  This  is  due  to  several  causes.  In  the  first  place, 
microscopic  objectives  have  been  naturally  constructed  to  give  the  clearest  image 
to  the  eye,  that  is  the  visual  image  as  it  is  sometimes  called,  is  for  microscopic  ob- 
servation, of  prime  importance.  The  actinic  or  photographic  image,  on  the  other 
hand,  is  of  prime  importance  for  photography.  For  the  majority  of  microscopic 
objects  transmitted  light  (  £  64)  must  be  used,  not  reflected  light  as  in  ordinary  vis- 
ion. Finally,  from  the  shortness  of  focus  and  the  smallness  of  the  lenses,  the 
proper  illumination  of  the  object  is  accomplished  with  some  difficulty,  and  the 
fact  of  the  lack  of  sharpness  over  the  whole  field  with  any  but  the  lower  powers, 
have  combined  to  make  photo-micrography  less  successful  than  ordinary  macro- 
photography.  So  tireless,  however,  have  been  the  efforts  of  those  who  believed  in 
the  ultimate  success  of  photo-micrography,  that  no%v  the  ordinary  achromatic  ob- 
jectives with  ortho-chromatic  or  isochromatic  plates  and  a  color  screen  or  petrol- 
eum light  give  good  results,  while  the  apochromatic  objectives  with  projection 
oculars  give  excellent  results,  even  in  hands  not  especially  skilled.  The  problem 
of  illumination  has  also  been  solved  by  the  construction  of  achromatic  and  apoch- 
romatic condensers  and  by  the  electric  and  other  powerful  lights  now  available. 
There  still  remains  the  difficulty  of  transmitted  light  and  of  so  preparing  the 
object  that  structural  details  stand  out  with  sufficient  clearness  to  make  a  picture 
which  approaches  in  definiteness  the  drawing  of  a  skilled  artist. 

The  writer  would  advise  all  who  wish  to  undertake  photo-micrography  seri- 
ously, to  study  samples  of  the  best  work  that  has  been  produced.  Among  those 
who  showed  th,e  possibilities  of  photo-micrographs  was  Col.  Woodward  of  the  U. 


fin  a  most  interesting  paper  by  A.  C.  Mercer  on  "The  Indebtedness  of  Pho- 
tography to  Microscopy,"  Photographic  Times  Almanac,  1887,  it  is  shown  that :  "To 
briefly  recapitulate,  photography  is  apparently  somewhat  indebted  to  microscopy 
for  the  first  fleeting  pictures  of  Wedgewood  and  Davy  [1802],  the  first  methods  of 
producing  permanent  paper  prints  [Reede,  1837-1839],  the  first  offering  of  prints 
for  sale,  the  first  plates,  engraved  after  photographs  for  the  purpose  of  book  illus- 
tration [Donne  &  Foucalt,  1845],  the  photographic  use  of  collodion  [Archer  &  Dia- 
mond, 1851],  and  finally,  wholly,  indebted  for  the  origin  of  the  gelatino-bromide 
process,  greatest  achievement  of  them  all  [Dr.  R.  L.  Maddox,  1871].  See  further 
for  the  history  of  Photo-micrography,  Neuhauss,  also  Bousfield. 


222 


PHO  TO-M1CROGRAPHY 


[CM.  VIII 


S.  Army  Medical  Museum.  The  photo-micrographs  made  by  him  and  exhibited 
at  the  Centennial  Celebration  at  Philadelphia  in  1876,  serve  still  as  models,  and  no 
one  could  do  better  than  to  study  them  and  try  to  equal  them  in  clearness  and 
general  excellence.  According  to  the  writer's  observation  no  photo-micrographs 
of  histological  objects  have  ever  exceeded  those  made  by  Woodward,  and  most  of 
them  are  vastly  inferior.  It  is  gratifying  to  state,  however,  that  at  the  present 
time  many  original  papers  are  partly  or  wholly  illustrated  by  photo-micrographs, 
and  no  country  has  produced  works  with  photo-micrographic  illustrations  superior 
to  those  in  "Wilson's  Atlas  of  Fertilization  and  Karyokinesis"  and  "Starr's  Atlas 
of  Nerve  Cells,"  issued  by  the  Columbia  University  Press. — 

In  passing  the  writer  would  like  to  pay  a  tribute  to  Mr.  W.  H.  Walmsley  who 
has  labored  in  advancing  photo-micrography  for  the  last  twenty  years.  His  con- 
venient apparatus  and  abundant  experience  have  been  placed  freely  at  the  com- 
mand of  every  interested  worker,  ;and  many  a  beginner  has  been  helped  over 
difficulties  by  him.  His  last  contribution  in  "International  Clinics,"  vol.  i.  ser. 
u,  12,  is  encouraging  in  the  highest  degree  both  for  its  matter  and  for  the 
illustrations. 


FIG.  183.  Zeiss'  Vertical  Photo-micro- 
graphic  Camera.  A.  Set  screw  holding  the 
rod  (S )  in  any  desired  position.  P,  Q.  Set 
screws,  by  which  the  bellows  are  held  in  place. 
B.  Stand  with  tripod  base  in  which  the  sup- 
porting rod  (S)  is  held.  This  rod  is  now 
graduated  in  centimeters  and  is  a  ready 
means  of  determining  the  length  of  the  cam- 
era. M.  Mirror  of  the  microscope.  L.  The 
sleeve  serving  to  make  a  light-tight  connection 
between  the  camera  and  microscope.  O.  The 
lower  end  of  the  camera.  R.  The  upper  end 
of  the  camera  where  the  focusing  screen  and 
plate  holder  are  situated.  (From  Zeiss"1  Photo- 
m  icrograph  ic  Ca  ta  log ) . 


As  the  difficulties  of  photo-micrography  are  so  much  greater  than  of  ordinary 
photography,  the  advice  is  almost  universal  that  no  one  should  try  to  learn  photo- 
graphy andtphoto-micrography  at  the  same  time,  but  that  one  should  learn  the 


CH.  K//7]  PHOTO-MICROGRAPHY  223 

processes  of  photography  by  making  portraits,  landscapes,  copying  drawings,  etc., 
and  then  when  the  principles  are  learned  one  can  take  up  the  more  difficult  subject 
of  photo-micrography  with  some  hope  of  success. 

The  advice  of  Sternberg  is  so  pertinent  and  judicious  that  it  is  reproduced  : 
"Those  who  have  had  no  experience  in  making  photo-micrographs  are  apt  to  ex- 
pect too  much  and  to  underestimate  the  technical  difficulties.  Objects  which 
under  the  microscope  give  a  beautiful  picture,  which  we  desire  to  reproduce  by 
photography  may  be  entirely  unsuited  for  the  purpose.  In  photographing  with 
high  powers  it  is  necessary  that  the  objects  to  be  photographed  be  in  a  single  plane 
and  not  crowded  together  and  overlying  each  other.  For  this  reason  photograph- 
ing bacteria  in  sections  presents  special  difficulties  and  satisfactory  results  can  only 
be  obtained  when  the  sections  are  extremely  thin  and  the  bacteria  well  stained. 
Even  with  the  best  preparations  of  this  kind  much  care  must  be  taken  in  selecting 
a  field  for  photography.  It  must  be  remembered  that  the  expert  microscopist,  in 
examining  a  section  with  high  powers,  has  his  finger  on  the  fine  adjustment  screw 
and  focuses  up  and  down  to  bring  different  planes  into  view.  He  is  in  the  habit  of 
fixing  his  attention  on  the  part  of  the  field  which  is  in  focus  and  discarding  the 
rest.  But  in  a  photograph  the  part  of  the  field  not  in  focus  appears  in  a  promi- 
nent way  which  mars  the  beauty  of  the  picture. ' ' 

APPARATUS    FOR   PHOTO-MICROGRAPHY 

|  362.  Camera. — For  the  best  results  with  the  least  expenditure  of  time  one  of 
the  cameras  especially  designed  for  photo-micrography  is  desirable  but  is  not  by 
any  means  indispensable  for  doing  good  work.  An  ordinary  photographic  camera, 
especially  the  kind  known  as  a  copying  camera,  will  enable  one  to  get  good  results, 
but  the  trouble  is  increased,  and  the  difficulties  are  so  great  at  best,  that  one  would 
do  well  to  avoid  as  many  as  possible  and  have  as  good  an  outfit  as  can  be  afforded 
(Figs.  184,  192). 

The  first  thing  to  do  is  to  test  the  camera  for  the  coincidence  of  the  plane  occu- 
pied by  the  sensitive  plate  and  the  ground  glass  or  focusing  screen.  Cameras  even 
from  the  best  makers  are  not  always  correctly  adjusted.  By  using  a  straight  edge 
of  some  kind,  one  can  measure  the  distance  from  the  inside  or  ground  side  of  the 
focusing  screen  to  the  surface  of  the  frame.  This  should  be  done  all  around  to  see 
if  the  focusing  screen  is  equally  distant  at  all  points  from  the  surface  of  the 
frame.  If  it  is  not  it  should  be  made  so.  When  the  focusing  screen  has  been  ex- 
amined, an  old  plate,  but  one  that  is  perfectly  flat,  should  be  put  into  the  plate 
holder  and  the  slide  pulled  out  and  the  distance  from  the  surface  of  the  plate 
holder  determined  exactly  as  for  the  focusing  screen.  If  the  distance  is  not  the 
same  the  position  of  the  focusing  screen  must  be  changed  to  correspond  with  that 
of  the  glass  in  the  plate  holder,  for  unless  the  sensitive  surface  occupies  exactly 
the  position  of  the  focusing  screen  the  picture  will  not  be  sharp,  no  matter  how7 
accurately  one  may  focus.  Indeed,  so  necessary  is  the  coincidence  of  the  plane  of 
the  focusing  screen  and  sensitive  surface  that  some  photo-micrographers  put  the 
focusing  screen  in  the  plate  holder,  focus  the  image  and  then  put  the  sensitive 
plate  in  the  holder  and  make  the  exposure  (Cox).  This  would  be  possible  with 
the  older  forms  of  plate  holders,  but  not  with  the  double  plate  holders  mostly  used 
at  the  present  day. 


224  PHOTO-MICROGRAPHY  \CH.  VIII 

\  3623.  Size  of  Camera. — The  majority  of  photo-micrographs  do  not  exceed  8 
centimeters  in  diameter  and  are  made  on  plates  Sx  u,  lox  13  or  13  x  18  centimeters 
(3^x4^  in.,  4x5  in.,  or  5x7  in.).  Most  of  the  vertical  cameras  are  for  plates  not 
exceeding  lox  13  centimeters  (4x5  in. )  but  Zeiss'  new  form  will  take  plates  21  x  21 
centimeters  (8^x8%"  in.). 

I  363.  Work  Room. — It  is  almost  self-evident  that  the  camera  must  be  in 
some  place  free  from  vibration.  Frequently  a  basement  room  where  the  camera 
table  may  rest  directly  on  the  cement  floor  or  on  a  pier  is  an  excellent  situation. 
Such  a  place  is  almost  necessary  for  the  best  work  with  high  powers.  For  those 
living  in  cities,  a  time  must  also  be  chosen  when  there  are  no  heavy  vehicles 
moving  in  the  streets.  For  less  difficult  work  an  ordinary  room  in  a  quiet  part  of 
the  house  or  laboratory  building  will  suffice. 

|  364.  Arrangement  and  Position  of  the  Camera  and  the  Microscope. — For 
much  of  photo-micrography  a  vertical  camera  and  microscope  are  to  be  preferred 
(Fig.  184).  Excellent  arrangements  were  perfected  long  ago,  especially  by  the 
French.  (See  Moitessier. ) 

Vertical  photo-micrographic  cam-eras  are  now  commonly  made,  and  by  some 
firms  only  vertical  cameras  are  produced.  They  are  exceedingly  convenient,  and 
do  not  require  so  great  a  disarrangement  of  the  microscope  to  make  the  picture  as 
do  the  horizontal  ones.  Van  Heurck  advises  their  use,  then  whenever  a  structure 
is  shown  with  especial  excellence  it  is  photographed  immediately.  The  variation 
in  size  of  the  picture  is  obtained  by  the  objective  and  the  projection  ocular  rather 
than  by  length  of  bellows  (see  below  Fig.  184).  It  must  not  be  forgotten,  how- 
ever, that  penetration  varies  inversely  as  the  square  of  the  power,  and  only  in- 
versely as  the  numerical  aperture  (§  34),  consequently  there  is  a  real  advantage  in 
using  a  low  power  of  great  aperture  and  a  long  bellows  rather  than  an  objective  of 
higher  power  with  a  short  bellows.  A  horizontal  camera  is  more  convenient  for  use 
with  the  electric  light  also  (Fig.  192). 

For  convenience  and  rapidity  of  work  a  microscope  with  mechanical  stage  is 
very  desirable.  It  is  also  an  advantage  to  have  a  tube  of  large  diameter  so  that 
the  field  will  not  be  too  greatly  restricted  (Fig.  189).  In  some  microscopes  the 
tube  is  removable  almost  to  the  nose-piece  to  avoid  interfering  with  the  size  of  the 
image.  The  substage  condenser  should  be  movable  on  a  rack  and  pinion.  The 
microscope  should  have  a  flexible  pillar  for  work  in  a  horizontal  position.  While  it 
is  desirable  in  all  cases  to  have  the  best  and  most  convenient  apparatus  that  is 
made,  it  is  not  by  any  means  necessary  for  the  production  of  excellent  work.  A 
simple  stand  with  flexible  pillar  and  good  fine  adjustment  will  answer. 

$  365.  Objectives  and  Oculars  for  Photo-Micrography. — The  belief  is  almost 
universal  that  the  apochromatic  objectives  are  most  satisfactory  for  photography. 
They  are  employed  for  this  purpose  with  a  special  projection  ocular.  Two  very 
low  powers  are  used  without  any  ocular  (Fig.  180).  Some  of  the  best  work 
that  has  ever  been  done,  however,  was  done  with  achromatic  objectives  (work  of 
Woodward  and  others).  One  need  not  desist  from  undertaking  photo-micrography 
if  he  has  good  achromatic  objectives.  From  a  somewhat  extended  series  of  ex- 
periments with  the  objectives  of  many  makers  the  good  modern  achromatic  ob- 
jectives were  found  to  give  excellent  results  when  used  without  an  ocular.  Most 
of  them  also  gave  good  results  with  projection  oculars,  although  it  must  be  said 
that  the  best  results  were  obtained  with  the  apochromatic  objectives  and  projec- 


FIG.  184. —  Vertical  photo- 
micrographic  camera, screen  and 
small  table.  The  table  is  about 
centimeters  high  and  in  the 
legs  are  large  screw  eyes  for 
leveling  screws.  The  operator 
can  stand  on  the  floor  and  per- 
form all  the  necessary  opera- 
tions, and  in  adjusting  the  mi- 
croscope can  sit  on  a  low  stool. 
The  screen  is  of  zinc  and 
has  two  heavy  lead  feet  to  hold 
it  steady.  Near  the  lower  left 
hand  corner  of  the  screen  is  an 
aperture  for  the  light  to  shine 
through  upon  the  mirror.  This 
opening  is  closed  by  a  black  slide 
which  is  just  balanced  so  that  it 
stays  in  any  position.  In  mak- 
ing the  exposure  it  is  raised 
sufficiently  to  admit  the  light  to 
the  mirror,  but  the  stage  is  left 
in  shadow.  This  screen  shades 
the  microscope  and  the  face  of 
the  operator.  ( Trans. 
Micr.  Soc.  1901. 


226 


PHO  TO-MICROGRAPHY 


[  CH.  VIII 


tion  oculars.  It  does  not  seem  to  require  so  much  skill  to  get  good  results  with 
the  apochromatics  as  with  the  achromatic  objectives.  The  majority  of  photo- 
micrographers  do  not  use  the  Huygenian  oculars  in  photography,  although  excel- 
lent results  have  been  obtained  with  them.  An  amplifier  is  sometimes  used  in 
place  of  an  ocular.  Considerable  experience  is  necessary  in  getting  the  proper 
mutual  position  of  objective  and  amplifier.  The  introduction  of  oculars 
especially  designed  for  projection,  has  led  to  the  discarding  of  ordinary  oculars 
and  of  amplifiers.  However  the  projection  oculars  of  Zeiss  restrict  the  field  very 
greatly,  hence  the  necessity  of  using  the  objective  alone  for  large  specimens.* 


No.  a. 


FIG.  185.  Projection  Oculars  with  section 
removed  to  show  the  construction.  Below  are 
shown  the  upper  end  with  graduated  circle  to 
indicate  the  amount  of  rotation  found  necessary 
to  focus  the  diaphragm  on  the  screen.  No.  2, 
No.  4.  The  numbers  indicate  the  amount  the 
ocular  magnifies  the  image  formed  by  the  ob- 
jective as  with  the  compensation  oculars.  (Zeiss' 
Catalog. ) 

$  366.  Difference  of  Visual  and  Actinic 
Foci. — Formerly  there  was  much  difficulty  ex- 
perienced in  photo-micrographing  on  account 
of  the  difference  in  actinic  and  visual  foci. 
Modern  objectives  are  less  faulty  in  this  respect 
and  the  apochromatics  are  practically  free 
from  it.  Since  the  introduction  of  orthochromatic  or  isochromatic  plates  and,  in 
many  cases  the  use  of  colored  screens,  but  little  trouble  has  arisen  from  differences 
in  the  foci.  This  is  especially  true  when  mono-chromatic  light  and  even  when 
petroleum  light  is  used.  In  case  the  two  foci  are  so  unlike  in  an  objective,  it 
would  be  better  to  discard  it  for  photography  altogether,  for  the  estimation  of  the 
proper  position  of  the  sensitive  plate  after  focusing  is  only  guess  work  and  the 
result  is  mere  chance.  If  sharp  pictures  cannot  be  obtained  with  an  objective 
when  petroleum  light  and  orthochromatic  plates  are  used  the  fault  may  not  rest 
with  the  objective  but  with  the  plate  holder  and  focusing  screen.  They  should  be 
very  carefully  tested  to  see  if  there  is  coincidence  in  position  of  the  focusing 
screen  and  the  sensitive  film  as  described  in  \  362. 

§367.  Apparatus  for  Lighting.— For  low  power  work  (35  mm.  and  longer 
focus)  and  for  large  objects,  some  form  of  bull's  eye  condenser  is  desirable 
although  fairly  good  work  may  be  done  with  diffused  light  or  lamp-light  reflected 
by  a  mirror.  If  a  bull's  eye  is  used  it  should  be  as  nearly  achromatic  as  possible. 
The  engraving  glass  shown  in  Fig.  188  answers  well  for  large  objects.  For  smaller 


*A  comparative  study  both  with  projection  oculars,  and  without  an  ocular 
was  made  with  the  achromatic  objective  25  mm.  (i  inch),  18  mm.  (finch),  5 
mm.  (\  to  \  inch)  and  2  mm.  (TV  inch)  homogeneous  immersion  of  the  Bausch  & 
Lomb  Optical  Co.;  Gundlach  Optical  Co.;  Leitz  ;  Reichert ;  Winkel,  Zeiss  and  the 
Spencer  Lens  Co.  Good  results  were  obtained  with  all  of  these  objectives  both 
with  and  without  projection  oculars. 


CH.  VIII] 


PHO  TO-MICROGRAPH  Y 


227 


objects  a  Steinheil  lens  combination  gives  a  more  brilliant  light  and  one  also  more 
nearly  achromatic.  For  high  power  work  all  are  agreed  that  nothing  will  take  the 
place  of  an  achromatic  condenser.  This  may  be  simply  an  achromatic  condenser, 
but  preferably  it  should  be  an  apochromatic  condenser.  Whatever  the  form  of  the 
condenser  it  should  possess  diaphragms  so  that  the  aperture  of  the  condenser  may 
be  varied  depending  upon  the  aperture  of  the  objective.  For  a  long  time  objec- 
tives have  been  used  as  achromatic  condensers,  and  they  are  very  satisfactory, 
although  less  convenient  than  a  special  condenser  whose  aperture  is  great  enough 
for  the  highest  powers  and  capable  of  being  reduced  by  means  of  diaphragms  to  the 
capacity  of  the  lower  objectives.  It  should  also  be  capable  of  accurate  centering. 


FIG.  186.     Arrangement  for  Artificial  Illumination. 

1.  Lamp  with  metal  chimney,  easily  made  by  rolling  up  some  ferrotype  plate 
and  making  a  slit-like  opening  in  one  side.     This  opening  should  be  covered  by  an 
oblong  cover-glass.  A  glass  slide,  being  of  considerable  thickness,  breaks  too  easily. 
The  lamp  should  have  a  wick  about  40  mm.  wide,  so  that  the  thickness  of  the  flame, 
if  taken  edgewise,  will  give  an  intense  light.     A  wide  flame  also  enables  one  to  get 
a  larger  image  of  the  flame,  and  thus  to  illuminate  a  larger  object  than  as  though  a 
small  flame  was  used. 

2.  BuWs-eye  condenser  on  a  separate  stand.     The  engraving  glass  shown  in 
Fig.  188,  or  the  tripod  magnifier  (Fig.  172}  answers  fairly .     The  Steinheil  lenses 
are  still  better. 

j.     Screen  showing  image  of  the  flame  inverted. 
The  lamp  and  bull's-eye  stand  are  on  blocks  with  screw-eyes  as  leveling  screws. 

$  368.  Objects  Suitable  for  Photo-micrographs. — While  almost  any  large  ob- 
ject may  be  photographed  well  with  the  ordinary  camera  and  photographic  objec- 
tive, only  a  small  part  of  the  objects  mounted  for  microscopic  study  can  be  photo- 
micrographed  satisfactorily.  Many  objects  that  give  beautiful  images  when  look- 
ing into  the  microscope  and  constantly  focusing  with  the  fine  adjustment,  appear 
almost  without  detail  on  the  screen  of  the  photo-micrographic  camera  and  in  the 
photo-micrograph. 


228 


PHO  TO-MICROGRAPHY 


[  CH.  V11I 


FIG.  187.  Adjustable  lens  holder.  This  lens  holder  will  take  magnifiers  of 
various  sizes,  and  from  its  adjustable  mechanism  is  very  convenient  for  dissecting, 
or  for  holding  a  Steinheil  and  other  lenses  for  illumination  (  The  Bausch  &  Lomb 
Opt.  Co.). 


FIG.  1 88.  Engraving  glass  to 
serve  as  a  condenser  and  for  a  dis- 
secting lens.  {Bausch  &  Lomb  Opt. 
Co.] 


If  one  examines  a  series  of  photo-micrographs  the  chances  are  that  the  greater 
number  will  be  of  diatoms,  plant  sections  or  preparations  of  insects.  That  is,  they 
are  of  objects  having  sharp  details  and  definite  outlines,  so  that  contrast  and  defi- 
niteness  may  be  readily  obtained.  Stained  microbes  also  furnish  favorable  objects 
when  mounted  as  cover-glass  preparations. 


CH.    VIII}  PHOTO-MICROGRAPHY  229 

Preparations  in  animal  histology  must  approximate  as  nearly  as  possible  to  the 
conditions  more  easily  obtained  with  vegetable  preparations.  That  is,  they  must 
be  made  so  thin  and  be  so  prepared  that  the  cell  outlines  will  have  something  of 
the  definiteness  of  vegetable  tissue.  It  is  useless  to  expect  to  get  a  clear  photo- 
graph of  a  section  in  which  the  details  are  seen  with  difficulty  when  studying  it 
under  the  microscope  in  the  ordinary  way. 

Many  sections  which  are  unsatisfactory  as  wholes,  may  nevertheless  have  parts 
in  which  the  structural  details  show  with  satisfactory  clearness.  In  such  a  case  the 
part  of  the  section  showing  details  satisfactorily  should  be  surrounded  by  a  delicate 
ring  by  means  of  a  marker  (see  Figs.  61-66).  If  one's  preparations  have  been 
carefully  studied  and  the  special  points  in  them  thus  indicated,  they  will  be  found 
far  more  valuable  both  for  ordinary  demonstration  and  for  photography.  The 
amount  of  time  saved  by  marking  one's  specimens  can  hardly  be  overestimated. 
The  most  satisfactory  material  for  making  the  rings  is  shellac  colored  with  lamp- 
black. 

Ten  years  ago  many  histologic  preparations  could  not  be  satisfactorily  photo- 
graphed. But  now  with  improved  section  cutters,  better  staining  and  mounting 
methods,  and  with  the  color  screens  (\  356)  and  isochromatic  plates  (§355)  almost 
any  preparation  which  shows  the'  elements  clearly  when  looking  into  the  micro- 
scope can  be  satisfactorily  photographed.  Good  photographs  cannot,  however,  be 
obtained  from  poor  preparations. 

\  369.  Light. — The  strongest  available  light  is  sunlight.  That  has  the  defect  of 
not  always  being  available,  and  of  differing  greatly  in  intensity  from  hour  to  hour, 
day  to  day  and  season  to  season.  The  sun  does  not  shine  in  the  evening  when 
many  workers  find  the  only  opportunity  for  work.  Following  the  sunlight  the 
electric  light  is  the  most  intense  of  the  available  lights.  Then  come  magnesium, 
acetylene,  the  lime  light,  the  gas-glow  or  Wellsbach  light,  and  lastly,  petroleum 
light.  The  last  is  excellent  for  the  majority  of  low  and  moderate  power  work. 
And  even  for  2  mm.  homogeneous  immersion  objectives,  the  time  of  exposure  is 
not  excessive  for  many  specimens  (40  seconds  to  3  minutes).  This  light  is  also 
cheapest  and  most  available  and  has  the  advantage  of  being  somewhat  yellow,  and 
therefore  in  many  cases  makes  the  use  of  a  color  screen  unnecessary  if  one  uses 
isochromatic  plates.  Acetylene  light  is  excellent  and  may  be  used  where  the  arc 
light  is  not  available. 

A  lamp  with  flat  wick  about  40  mm.  (i^  in.)  wide  has  been  found  most  gen- 
erally serviceable.  For  large  objects  and  low  powers  the  flame  may  be  made 
large  and  the  face  turned  toward  the  mirror.  This  will  light  a  large  field.  For 
high  powers  the  edge  toward  the  mirror  gives  an  intense  light.  The  ordinary 
glass  chimney  answers  well,  especially  where  a  metal  screen  is  used  as  shown 
in  Fig.  184. 

EXPERIMENTS    IN   PHOTO-MICROGRAPHY 

§  370.  The  following  experiments  are  introduced  to  show  prac- 
tically just  how  one  would  proceed  to  make  photo-micrographs  with 
various  powers,  and  be  reasonably  certain  of  fair  success.  If  one  con- 
sults prints  or  the  published  figures  made  directly  from  photo-micro- 
graphs it  will  be  seen  that,  excepting  the  bacteria,  the  magnification 
ranges  mostly  between  10  and  150  diameters. 


230  PHOTO-MICROGRAPHY  [CH.  VIII 

§  371.  Focusing  Screen  for  Photo-Micrography. — One  cannot 
expect  a  picture  sharper  than  the  image  seen  on  the  focusing  screen. 
Hence  the  greatest  care  must  be  taken  in  focusing.  The  general  focus- 
ing may  be  done  with  the  unaided  eye  on  the  ground  glass,  but  for  the 
final  focusing  a  clear  screen  and  a  focusing  glass  must  be  used.  (Figs. 
172,  173).  See  §  347.  With  the  clear  focusing  screen  one  cannot  at 
first  see  the  image  without  using  a  focusing  glass,  but  with  a  little  ex- 
perience the  aerial  image  may  be  seen  as  with  the  microscope  (§  54). 

§  372.  Photo-micrographs  of  20  to  50  Diameters. — For  pic- 
tures under  15  or  20  diameters  it  is  better  to  use  the  camera  for  embryos 
with  the  objective  in  the  end  of  the  camera,  and  the  special  microscope 
stand  for  focusing  (Fig.  175). 

For  pictures  at  25  to  50  diameters  one  may  use  the  microscope  with 
a  low  objective,  25  to  35  mm.  equivalent  focus,  and  no  ocular  (Fig. 
184).  The  object  is  placed  on  the  stage  of  the  microscope,  and  focused 
as  in  ordinary  observation.  If  a  vertical  microscope  is  used  the  light 
from  the  petroleum  lamp  or  other  artificial  light,  is  reflected  upward  by 
the  mirror.  It  may  take  some  time  to  get  the  whole  field  lighted 
evenly.  Refer  back  to  §  95  for  directions.  In  some  cases  it  may  be 
advisable  to  discard  the  condenser  and  use  the  mirror  only.  For  some 
purposes  one  will  get  a  better  light  by  placing  the  bull's  eye  or  other 
condenser  between  the  lamp  and  the  mirror  to  make  the  rays  parallel 
or  even  to  make  a  sharp  image  of  the  lamp  flame  on  the  mirror.  Re- 
member also  that  in  many  cases  it  is  necessary  to  have  a  color  screen 
between  the  source  of  light  and  the  object  (§  356). 

For  a  horizontal  camera  it  is  frequently  better  to  swing  the  mirror 
entirely  out  of  the  way  and  allow  the  light  to  enter  the  condenser 
directly  or  after  traversing  the  bull's  eye  (Figs.  182,  186).  If  the  ob- 
ject is  small  an  achromatic  combination  like  a  Steinheil  magnifier  or  an 
engraving  glass  is  excellent  (Fig.  188).  When  the  light  is  satisfac- 
tory as  seen  through  an  ordinary  ocular,  remove  the  ocular. 

(A)  Photographing  without  an  Ocular. — After  the  removal  of  the 
ocular  put  in  the  end  of  the  tube  a  lining  of  black  velvet  to  avoid  re- 
flections. Connect  the  microscope  with  the  camera,  making  a  light- 
tight  joint  and  focus  the  image  on  the  focusing  screen.  One  may  make 
a  light-tight  connection  by  the  use  of  black  velveteen  or  more  con- 
veniently by  the  Zeiss'  double  metal  hood  which  slips  over  the  end  of 
the  tube  of  the  microscope,  and  into  which  fits  a  metal  cylinder  on  the 
lower  end  of  the  camera  (Figs.  184,  189,  183).  In  the  last  figure  the 
connection  has  been  made. 


CH.  VIII'} 


PHO  TO-MICROGRAPHY 


231 


FIG.  189.  Zeiss1  special  photo-micrographic  stand.  It  has  a  very  large  tube, 
a  slow  acting  fine  adjustment,  mechanical  stage  and  all  appliances  for  the  most  sat- 
isfactory work.  ( Cut  loaned  by  Eimer  and  Amend}. 

It  will  be  necessary  to  focus  down  considerably  to  make  the  image 
clear.  Lengthen  or  shorten  the  bellows  to  make  the  image  of  the  de- 
sired size,  then  focus  with  the  utmost  care.  In  case  the  field  is  too 
much  restricted  on  account  of  the  tube  of  the  microscope,  remove  the 
draw-tube.  When  all  is  in  readiness  it  is  well  to  wait  for  three  to  five 
minutes  and  then  to  see  if  the  image  is  still  sharply  focused.  If  it  has 
got  out  of  focus  simply  by  standing,  a  sharp  picture  could  not  be  ob- 


232  PHOTO-MICROGRAPHY  \_CH.  VIII 

FIGS.  190-191.  Fine  tint,  half-tone  reproductions  of  photo-micrographs  of  sec- 
tions made  by  Mrs.  Gage,  to  show  the  possibilities  of  photo-micrography  with  pho- 
tographic objectives  and  with  low  microscopic  objectives  without  a  projection  ocular. 

1.  Frontal  section   of  the  head  of  a  large  red  Diemyctylus  viridescens  (red 
newt]  at  the  level  of  the portae  of  the  brain,  magnified  10  diameters.     Negative 
made  with  a  Gundlach  perigraphic  objective  of  about  oo  mm.  equivalent  focus. 

2.  Frontal  section  of  a  larval  Diemyctylus  about  10  millimeters  in  length. 
Negative  made  with  a  Winkel  objective  of  22  millimeters  equivalent  focus ;  no 
ocular.     Magnified  50    diameters.     (Mrs.    Susanna    Phelps    Gage,    the     Wilder 
Quarter  Century  Book}. 

tained.  If  it  does  not  remain  in  focus,  something  is  faulty.  When 
the  image  remains  sharp  after  focusing  make  the  exposure.  From  20 
to  60  seconds  will  usually  be  sufficient  time  with  medium  plates  and 
the  light  as  described.  If  a  color  screen  is  used  it  will  require  40-300 
seconds,  i.  e.,  2  to  5  times  as  long,  for  a  proper  exposure  (§  359). 

B.  Photographing  with  a  Projection  Ocular. — If  the  object  is  small 
enough  to  be  included  in  the  field  of  a  projection  ocular  (Fig.  185)  use 
that  for  making  the  negative  as  follows  :  Swing  the  camera  around  so 
that  it  will  leave  the  microscope  free.  Use  an  ordinary  ocular,  focus 
and  light  the  object,  then  insert  a  projection  ocular  in  place  of  the  or- 
dinary one,  and  swing  the  camera  back  over  the  microscope.  It  is  not 
necessary  to  use  an  ordinary  ocular  for  the  first  focusing,  but  as  its 
field  is  larger  it  is  easier  to  find  the  part  to  be  photographed.  The 
first  step  is  then  to  focus  the  diaphragm  of  the  projection  ocular 
sharply  on  the  focusing  screen.  Bring  the  camera  up  close  to  the  mi- 
croscope and  then  screw  out  the  eye-lens  of  the  ocular  a  short  distance. 
Observe  the  circle  of  light  on  the  focusing  screen  to  see  if  its  edges  are 
perfectly  sharp.  If  not,  continue  to  screw  out  the  eye  lens  until  it  is. 
If  it  cannot  be  made  sharp  by  screwing  it  out  reverse  the  operation. 
Unless  the  edge  of  the  light  circle,  i.  e. ,  the  diaphragm  of  the  ocular, 
is  sharp,  the  resulting  picture  will  not  be  satisfactory. 

It  should  be  stated  that  for  the  X  2  projection  ocular  the  bellows 
of  the  camera  must  be  extended  about  30  or  40  centimeters  or  the 
diaphragm  cannot  be  satisfactorily  focused  on  the  screen.  The  X4 
projection  ocular  can  be  focused  with  the  bellows  much  shorter.  For 
either  projection  ocular  the  screen  distance  can  be  extended  almost 
indefinitely. 

When  the  diaphragm  is  sharply  focused  on  the  screen,  the  micro- 
scope is  focused  as  though  no  ocular  were  present,  that  is,  first  with  the 
unaided  eye  then  with  the  focusing  glass.  The  exposure  is  also  made 
in  the  same  way,  although  one  must  have  regard  to  the  greater  mag- 


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CH.  VIII}  PHOTO-MICROGRAPHY  233 

nification  produced  by  the  projection  ocular  and  increase  the  time  ac- 
cordingly ;  thus  when  the  X  4  ocular  is  used,  the  time  should  be  at 
least  doubled  over  that  when  no  ocular  is  employed.  The  time  will  be 
still  further  increased  if  a  color  screen  is  used  (§  359). 

Zeiss  recommends  that  when  the  bellows  have  sufficient  length  the 
lower  projection  oculars  be  used,  but  with  a  short  bellows  the  higher 
ones.  It  is  also  sometimes  desirable  to  limit  the  size  of  the  field  by 
putting  a  smaller  diaphragm  over  the  eye  lens.  This  also  aids  in 
making  the  field  uniformly  sharp. 

§373.  Determination  of  the  Magnification  of  the  Photo- 
Micrograph. — After  a  successful  negative  has  been  made,  it  is  desirable 
and  important  to  know  the  magnification.  This  is  easily  determined 
by  removing  the  object  and  putting  in  its  place  a  stage  micrometer. 
If  the  distance  between  two  or  more  of  the  lines  of  the  image  on 
the  focusing  screen  is  obtained  with  dividers  and  the  distance  meas- 
ured on  one  of  the  steel  rules,  the  magnification  is  found  by  dividing 
the  size  of  the  image  by  the  known  size  of  the  object  (§  154).  If  now 
the  length  of  the  bellows  from  the  tube  of  the  microscope  is  noted,  say 
on  a  record  table  like  that  in  section  360,  one  can  get  a  close  approxi- 
mation to  the  power  at  some  other  time  by  using  the  same  optical  com- 
bination and  length  of  bellows. 

For  obtaining  the  magnification  at  which  negatives  are  made  it  is 
a  great  advantage  to  have  one  micrometer  in  half  millimeters  ruled 
with  coarse  lines  for  use  with  the  lower  powers,  and  one  in  o.  i  and 
o.oi  millimeter  ruled  with  fine  lines  for  the  higher  powers. 

§  374.  Photo-Micrographs  at  a  Magnification  of  100  to  150 
Diameters. — For  this,  the  simple  arrangements  given  in  the  preceding 
section  will  answer,  but  the  objectives  must  be  of  shorter  focus,  8  to  3 
mm.  It  is  better,  however,  to  use  an  achromatic  condenser  instead  of 
the  engraving  glass  or  the  Steinheil  lens. 

§  375.  Lighting  for  Photo-Micrography  with  Moderate  and 
High  Powers. — (100  to  2,500  diameters).  No  matter  how  good  one's 
apparatus,  successful  photo-micrographs  cannot  be  made  unless  the  ob- 
ject to  be  photographed  is  properly  illuminated.  The  beginner  can  do 
nothing  better  than  to  go  over  with  the  greatest  care  the  directions  for 
centering  the  condenser,  for  centering  the  source  of  illumination,  and 
the  discussion  of  the  proper  cone  of  light  and  lighting  the  whole  field, 
as  given  on  pp.  41-52.  Then  for  each  picture  the  photographer  must 
take  the  necessary  pains  to  light  the  object  properly.  An  achromatic 
condenser  is  almost  a  necessity  (§  80).  Whether  a  color-screen  should 


234  PHOTO-MICROGRAPHY  \_CH.  VIII 

be  used  depends  upon  judgment  and  that  can  be  attained  only  by  ex- 
perience. In  the  beginning  one  may  try  without  a  screen,  and  with 
different  screens  and  compare  results. 

A  plan  used  by  many  skilled  workers  is  to  light  the  object  and  the 
field  around  it  well  and  then  to  place  a  metal  diaphragm  of  the  proper 
size  in  the  camera  very  close  to  the  plate  holder.  This  will  insure  a 
clean,  sharp  margin  to  the  picture.  This  metal  diaphragm  must  be 
removed  while  focusing  the  diaphragm  of  the  projection  ocular,  as  the 
diaphragm  opening  is  smaller  than  the  image  of  the  ocular  dia- 
phragm. 

If  the  young  photo-micrographer  will  be  careful  to  select  for  his 
first  trials,  objects  of  which  really  good  photo- micrographs  have 
already  been  made,  and  then  persists  with  each  one  until  fairly  good 
results  are  attained,  his  progress  will  be  far  more  rapid  than  as  if  poor 
pictures  of  many  different  things  were  made.  He  should,  of  course, 
begin  with  low  magnifications. 

§  376.  Adjusting  the  Objective  for  Cover-Glass.— After  the 
object  is  properly  lighted,  the  objective,  if  adjustable,  must  be  cor- 
rected for  the  thickness  of  cover.  If  one  knows  the  exact  thickness 
of  the  cover  and  the  objective  is  marked  for  different  thicknesses,  it  is 
easy  to  get  the  adjustment  approximately  correct  mechanically,  then 
the  final  corrections  depend  on  the  skill  and  judgment  of  the  worker. 
It  is  to  be  noted  too  that  if  the  objective  is  to  be  used  without  a  projec- 
tion ocular  the  tube-length  is  practically  extended  to  the  focusing  screen 
and  as  the  effect  of  lengthening  the  tube  is  the  same  as  thickening  the 
cover- glass,  the  adjusting  collar  must  be  turned  to  a  higher  number 
than  the  actual  thickness  of  the  cover  calls  for  (see  §  103). 

§  377.  Photographing  Without  an  Ocular. — Proceed  exactly 
as  described  for  the  lower  power,  but  if  the  objective  is  adjustable  make 
the  proper  adjustment  for  the  increased  tube-length  (§  103). 

§  378.  Photographing  with  a  Projection  Ocular. — Proceed  as 
described  in  §  372  B,  only  in  this  case  the  objective  is  not  to  be  adjusted 
for  the  extra  length  of  bellows.  If  it  is  corrected  for  the  ordinary 
ocular,  the  projection  ocular  then  projects  this  correct  image  upon  the 
focusing  screen. 

§  379.  Photo-Micrographs  at  a  Magnification  of  500  to  2000 
Diameters. — For  this  the  homogeneous  immersion  objective  is  em- 
ployed, and  as  it  requires  a  long  bellows  to  get  the  higher  magnifica- 
tion with  the  objective  alone,  it  is  best  to  use  the  projection  oculars. 


CH.  VIII}  PHOTO-MICROGRAPHY  235 

For  this  work  the  directions  given  in  §  372  B  must  be  followed  with 
great  exactness.  The  edge  of  the  petroleum  lamp  flame  is  sufficient  to 
fill  the  field  in  most  cases.  With  many  objects  the  time  required  with 
good  lamp  light  is  not  excessive  ;  viz.,  40  seconds  to  3  minutes.  The 
reason  of  this  is  that  while  the  illumination  diminishes  directly  as  the 
square  of  the  magnification,  it  increases  with  the  increase  in  numerical 
aperture,  so  that  the  illuminating  power  of  the  homogeneous  immersion 
is  great  in  spite  of  the  great  magnification  (§  34). 

For  work  with  high  powers  a  stronger  light  than  the  petroleum 
lamp  is  employed  by  those  doing  considerable  photo-micrography. 
Good  work  may  be  done,  however,  with  the  petroleum  lamp. 

It  may  be  well  to  recall  the  statement  made  in  the  beginning,  that 
the  specimen  to  be  photographed  must  be  of  especial  excellence  for  all 
powers.  No  one  will  doubt  the  truth  of  the  statement  who  undertakes 
to  make  photo-micrographs  at  a  magnification  of  500  to  2000  diameters. 

If  one  has  a  complete  outfit  with  electric  arc  light  (Fig.  192)  the 
time  required  for  photographing  objects  is  much  reduced,  i.  e.  ranging 
from  i  to  20  seconds  even  with  a  color  screen.  As  the  light  is  so  in- 
tense with  the  arc  light  it  is  necessary  to  soften  it  greatly  for  focusing. 
Several  thicknesses  of  ground  glass  placed  between  the  lamp  and  the 
microscope  will  answer.  These  are  removed  before  taking  the  nega- 
tive. It  is  well  also  to  have  a  water  bath  on  the  optical  bench  to  ab- 
sorb the  heat  rays.  This  should  be  in  position  constantly  (see  Ch.  IX). 

§380.  Use  of  Oculars  in  Photo-Micrography. — There  is  much 
diversity  of  opinion  whether  or  not  the  ordinary  oculars  used  for  ob- 
servation should  be  used  in  photographing.  Excellent  results  have 
been  obtained  with  them  and  also  without  them. 

For  great  magnification  Zeiss  recommends  the  use  of  the  compen- 
sation oculars  with  the  apochromatics. 

The  Zeiss  projection  oculars  may  be  used  with  achromatic  objectives 
of  large  aperture  as  well  as  with  the  apochromatics. 

PHOTOGRAPHING   OPAQUE    OBJECTS   AND   METALLIC   SURFACES 
WITH    A   MICROSCOPE 

All  of  the  objects  cansidered  in  the  first  part  of  this  chapter  are  opaque  and 
some  of  them  were  to  be  photographed  somewhat  larger  than  natural  size.  To 
meet  the  needs  of  modern  work,  especially  with  metals  and  alloys  one  must  be 
able  to  examine  and  photograph  prepared  surfaces  at  magnifications  ranging  from 
five  or  ten  to  five  hundred  or  more  diameters. 

\  381.  Microscope  for  Opaque  Objects. — If  one  does  not  need  to  magnify 
more  than  about  100  diameters,  any  good  microscope  will  answer.  For  the  higher 


236 


PHO  TO  -MICROGRA  PHY 


\CH.  VIII 


CH.  VIII} 


PHO  TO-MICROGRA  PH  Y 


237 


FiG.  192.  Buxton's  Photo- Micrograp hie  outfit  for  use  with  the  arc  light. 
(Jour.  Ap.  Microscopy,  igoi,  p.  1367}.  (Cut  loaned  by  the  Bausch  &  Lomb.  Opt. 
Co.)  As  will  be  seen  from  the  figure  this  apparatus  is  for  work  in  the  horizontal 
Position.  The  optical  bench  containing  the  microscope,  water  bath,  color  screen  and 
the  electric  light,  swings  sidewise  sufficiently  for  the  operator  to  arrange  the  speci- 
men exactly  as  desired.  It  then  swings  back  into  position  and  is  joined  to  the 
camera.  This  is  in  two  sections  for  either  a  short  or  long  bellows.  This  seems  to 
be  the  most  convenient  of  all  the  expensive  outfits  for  photo-micrography . 

powers  it  is  far  more  convenient  to  employ  a  special  microscope  for  metallography 
(micro-metalloscope.)  (German,  Metallmikroskop ;  French,  Microscope  pour 
1' etude  des  surfaces  metalliques  et  des  objets  opaque).  (Fig.  193.) 


FIG.  193.  Special  microscope  of  the  Boston  Testing  Laboratories  for  the  study 
and  photography  of  metals  and  alloys  (\  381).  (Cut  loaned  by  the  Boston  Testing 
Laboratories. ) 


238  PHOTO-MICROGRAPHY  [CH.  VIII 

Such  a  microscope  has  the  following  general  characters  :  The  stage  is  mova- 
ble up  and  down  with  rack  and  pinion,  it  is  rotary  and  more  or  less  mechanical  by 
means  of  centering  screws.  With  some  at  least  the  stage  may  be  removed 
entirely.  No  substage  condenser  is  present,  and  a  mirror  is  only  present  for  occa- 
sional transparent  objects.  A  revolving  nose-piece  is  not  so  good  as  the  objective 
changers. 

§  382.  Illumination  of  Opaque  Objects. — (A)  for  25  to  100  diameters.  The 
directions  of  Mr.  Walmsley  are  excellent  (Trans.  Amer.  Micr.  Soc.,  1898,  p.  191). 
"Altogether  the  best  light  for  the  purpose  is  diffused  daylight.  Proper  lighting  is 
more  easily  obtained  with  a  vertical  camera.  An  even  illumination  avoiding  deep 
shadows  is  preferable  in  most  cases  and  is  more  easily  attained  with  the  object  in 
a  horizontal  position.  For  many  objects  it  is  better  not  to  use  a  bull's  eye  or  any 
form  of  condenser  but  for  others  the  condenser  may  be  needed,  but  when  the  con- 
denser is  used  one  must  avoid  too  much  glare.  The  now  little  used  parabolic  re- 
flector and  Lieberkiihn  serve  well  in  many  cases,  but  he  adds  "the  majority  yield 
better  results  under  the  most  simple  forms  of  illumination,"  i.  e.,  with  the  dif- 
fused light  from  the  window.  This  has  been  the  experience  of  the  writer  also. 

In  case  diffused  daylight  is  employed  the  camera  should  be  near  a  good  sized 
window,  and  the  object  should  be  somewhat  below  the  window  ledge  so  that  the 
illumination  is  partly  from  above  and  from  the  side.  (This  is  easily  attained  with 
the  small  table  and  vertical  camera  shown  in  Figs.  175,  184).  The  vertical  illum- 
inator is  advantageous  for  these  powers  also.  See  (B. ). 

(B)  For  100  to  500  diameters, — For  the  magnifications  above  50  it  is  desirable 
and  for  those  above  100  it  is  necessary  to  use  some  form  of  "vertical  illuminator," 
that  is  some  arrangement  by  which  the  light  is  reflected  down  through  the  objec- 
tive upon  the  object,  the  objective  acting  as  a  condenser,  and  from  the  object 
back  through  the  objective  and  ocular  to  the  eye  of  the  observer.  This  is  accom- 
plished in  two  ways  : 

1 i )  By  means  of  a  small  speculum-metal  mirror  in  the  tube  of    the  micro- 
scope.    This  is  set  at  an  angle  of  45  degrees  and  the  light  thrown  into  the  tube 
upon  it  is  reflected  straight  down  through  the   objective  upon   the  object.     The 
speculum  metal  being  opaque  cuts  out  a  part  of  the  light.     Instead  of  a  metal 
mirror  a  circular  disc  of  glass   is   now  more   frequently  used.     This   allows  the 
major  part  of  the  light  reflected  from  the  object,  up  through  the  objective  to 
reach  the  eye. 

( 2 )  By  means  of  a  small  glass  45  degree  prism  inserted  into  the  side  of  the 
objective  or  of  a  special  adapter.     The  light  is  from  the  side  of  the  microscope,  and 
is  reflected  by  the  prism  straight  down  through  the  objective  upon  the  object  as 
before.* 


*The  idea  of  the  vertical  illuminator  apparently  originated  with  Hamilton  L. 
Smith.  He  used  the  metal  reflector.  Beck  substituted  a  cover-glass  and  Powell 
and  L/ealand  a  disc  of  worked  glass;  i.  e.  glass  that  had  been  carefully  polished 
and  leveled  on  the  two  sides.  Carpenter-Dallinger,  pp.  336-338. 

The  use  of  the  prism  with  the  objective  is  due  to  Tolles  (see  Jour.  Roy.  Micr. 
Soc.,  vol.  iii,  1880,  pp.  526,  574). 

In  Zeiss'  catalog  the  prism  form  is  figured.  In  the  catalog  of  Nachet  both  the 
glass  disc  and  the  prism  forms  are  figured. 


CH.  VIII}  PHOTO-MICROGRAPHY  239 

For  both  these  devices  uncovered  objects  are  most  successful  or  if  the  object 
is  covered  it  must  be  in  optical  contact  with  the  cover-glass.  Naturally  good  re- 
flecting surfaces  like  the  rulings  on  polished  metal  bars  give  most  satisfactory 
images,  hence  this  method  of  illumination  is  especially  adapted  to  micro-metal- 
lography. Indeed,  without  some  such  adequate  method  of  illumination  the  study 
of  metals  and  alloys  with  high  powers  would  be  impossible.  So  successful  is  it 
that  oil  immersion  objectives  may  be  used.  ( Carpenter- Dallinger,  pp.  335-338). 
§  383.  Light  for  the  Vertical  Illuminator. — For  moderate 
powers  one  may  place  the  microscope  in  front  of  a  window,  or  one  ma}* 
use  a  petroleum  or  gas  lamp.  For  the  higher  powers  acetylene  or 
preferably  the  electric  arc  light  is  used.  In  either  case  it  may  be  neces- 
sary to  soften  the  light  somewhat  either  by  a  color  screen  or  by  some 
ground  glass.  The  light  should  be  concentrated  upon  the  exposed  end 
of  the  prism  or  into  the  hole  leading  to  the  glass  disc.  Both  the  prism 
and  the  disc  should  be  adjustable  for  different  objectives  and  different 
specimens.  The  cone  of  light,  especially  with  the  electric  arc  light, 
should  be  enclosed  in  a  hollow  metal  or  asbestos  cone  to  avoid  the  glare 
in  the  eyes  of  the  operator,  and  it  riiay  be  necessary  to  soften  the  light 
with  ground  glass  before  attempting  to  focus  and  arrange  the  speci- 
men. This  ground  glass  would  in  most  cases  be  removed  before  mak- 
ing the  exposure  (§  379). 

With  the  electric  light  and  for  long  exposure  or  observation  a 
water  bath  to  absorb  the  heat  rays  will  be  necessary  to  avoid  injuring 
the  lenses.  (See  also  under  projection  in  the  next  chapter). 

As  it  is  somewhat  difficult  to  adjust  the  light  in  a  way  to  give  the 
best  effect,  one  can  see  the  advantage  of  the  adjustment  for  raising  and 
lowering  the  stage.  This  will  serve  for  all  but  the  finest  focusing,  and 
thus  avoid  moving  the  tube  for  focusing  enough  to  throw  the  lighting 
out  of  adjustment.  It  might  be  advantageous  to  have  a  fine  adjust- 
ment on  the  stage  also. 

\  384.  Mounting  of  Objects.— For  observation  only  and  with  low  powers, 
the  objects  may  be  mounted  either  in  a  liquid  or  dry  as  seems  best.  There  should 
be  a  black  background  for  most  objects,  then  light  will  reach  the  eye  only  from 
the  object.  A  light  background  is  sometimes  desirable,  especially  where  one  cares 
only  for  outlines. 

§385.  Preparation  of  Metallic  Surfaces.— In  the  first  place  a  flat  face  is 
obtained  by  grinding  or  filing,  and  then  this  is  polished.  For  polishing,  finer 
and  finer  emery  or  other  polishing  powders  are  used,  (rouge  or  diamantine, 
or  specially  prepared  alumnina,  etc).  The  aim  is  to  get  rid  of  the  scratches  so 
that  the  surface  will  be  smooth  and  free  from  lines. 

\  386.  Etching. — After  the  surface  is  polished  it  should  be  etched  with  some 
substance.  This  etching  material  will  corrode  the  less  resistant  material,  the 
edges  of  crystals,  etc.,  so  that  the  structure  will  appear  clearly.  For  etching, 


240  PHOTO-MICROGRAPHY  [C//.  VI 7/ 

tincture  of  iodine,  nitric  acid  in  various  degrees  of  strength,  hydrochloric  acid, 
etc. ,  are  used  or  one  may  use  electricity,  the  metal  being  immersed  in  an  indiffer- 
ent liquid.  See  numerous  articles  in  the  Metallographist  for  methods  and  micro- 
graphs. 

After  the  etching,  the  surface  should  be  washed  well  with  water  to  remove  the 
etcher.  L/e  Chatelier  recommends  that  the  etched  surface  when  dry  be  coated 
with  a  very  thin  coating  of  collodion  to  avoid  tarnishing.  The  preparation  will 
then  last  for  several  months  untarnished. 

§  387.  Mounting  the  Preparations  of  Metal. — In  order  to  get  a  satisfactory 
image  the  flat,  polished  and  etched  face  should  be  at  right  angles  to  the  optic  axis. 
For  preliminary  observation  one  can  approximate  this  by  mounting  the  specimen 
on  a  piece  of  beeswax.  (Behrens).  Very  elaborate  arrangements  of  the  stage 
have  also  been  devised  ( Reichert) .  A  simple  and  effective  device  is  shown  in  Fig. 
193  in  which  the  specimen  is  held  against  the  under  side  of  the  plane  face  of  the 
stage  attachment.  Rubber  bands  answer  well  to  support  the  metal,  and  only  one 
side  need  be  flat. 

\  388.  'Photographing  Opaque  Objects. — The  general  directions  given  in  \  347 
should  be  followed  with  the  necessary  modifications.  The  time  of  exposure  is 
usually  considerably  greater  with  opaque  objects  than  with  transparent  ones. 
Very  few  such  objects  can  be  photgraphed  in  less  than  30  seconds,  even  with  day- 
light For  metallic  surfaces  and  magnifications  of  100,  150,  250  to  500,  with  the 
electric  arc  light  as  illuminant  the  time  required  for  favorable  objects  is  i,  2, 
4  and  7  seconds  ;  with  the  Wellsbach  lamp  the  time  is  5,  10,  30  and  60  minutes 
(Sauveur). 


FIG.  194.     Rack  for   drying  negatives 
(Rochester  Opt.  Co}. 


FIG.  194. 

References  to  Ch.  VIII. 

See  the  works  and  journals  dealing  with  photography. 

For  Photo-Micrography  see  Pringle,  Bousfield,  Neuhauss,  Sternberg,  Francotte 
and  the  special  catalogs  on  photo-micrography  and  projection  issued  by  the  great 
opticians.  The  Journal  of  the  Royal  Microscopical  Society  and  of  the  Quekett 
Micr.  Club  ;  Zeit.  wiss.  Mikroskopie  ;  the  Trans  Amer.  Micr.  Soc. ;  the  Amer. 
Monthly  Micr.  Journal  ;  the  Journal  of  Applied  Microscopy. 

For  the  photography  of  metallic  surfaces,  see  the  various  journals  of  engineer- 
ing and  metallurgy,  but  especially  Sauveur's  journal,  the  Metallographist,  begun 
in  1898. 


CH.  VIII}  PHOTO-MICROGRAPHY  241 

ENLARGEMENTS  ;    LANTERN    SLIDES  ;    PHOTOGRAPHING 
BACTERIAL    CULTURES 

g  389.  Enlargements. — As  a  low  power  objective  has  greater  depth  of  focus 
or  penetration  than  a  higher  power  (\  34),  it  is  desirable  in  many  cases  to  make  a 
negative  of  an  object  with  considerable  depth  at  a  low  magnification,  and  then 
to  enlarge  this  picture  to  the  desired  size.  As  a  rule  negatives  will  not  bear  an 
enlargement  of  more  than  five  diameters. 

For  this  work  the  camera  shown  in  Fig.  181  is  excellent,  and  the  special  mi- 
croscope stand  shown  in  this  figure  and  in  Fig.  175  serves  to  enable  one  to  get  a 
very  exact  focus. 

One  must  select  an  objective  for  the  enlargement  with  a  field  of  sufficient  size 
to  cover  the  part  of  the  negative  to  be  enlarged.  An  objective  of  60  to  100  mm. 
focus  will  answer  in  most  cases. 

For  the  illumination  the  camera  can  be  elevated  against  the  sky,  or  artificial 
light  may  be  used.  It  is  not  easy  to  light  so  large  a  surface  evenly  by  artificial 
light. 

(A)  Enlargement  on  Bromide  Paper. — For  this  the  negative  is  put  in  place 
and  by  pulling  out  the  bellows  the  proper  amount,  one   gets   the  right  magnifica- 
tion.    Focus  now  as  for  any  other  object,  using  the  fine  adjustment  and  focusing 
glass. 

For  great  exactness  one  must  put  a  clear  glass  in  the  plate  holder  and  focus 
on  the  surface  away  from  the  objective.  Then  place  the  bromide  paper  on  this 
clear  glass  and  put  another  over  it  to  hold  it  flat  against  the  first  plate  of  glass. 
The  sensitive  surface  will  then  be  in  the  exact  plane  of  the  focus  and  the  picture 
will  be  sharp. 

For  the  development  and  subsequent  treatment  of  the  paper,  follow  the 
directions  of  the  makers. 

(B)  Enlargement  on  a  Glass  Plate. — One  may  proceed  in  enlarging  as  for 
making  lantern  slides  and  make  a  positive  on  a  glass  plate.     If  it  is  then  desired 
to  get  a  negative   for  printing,  place  this  positive  on  the  microscope  stand  and 
make  a  negative  from  it  as  if  it  were  an  object.     Or  one  may  make  a  contact  im- 
pression as  is  frequently  done  in  lantern  slide  making.     By  this  method  one  must 
make  three  separate  pictures,  (i)the  original  photo-micrographic  negative  ;  (2) 
the  enlarged  positive  from  this  ;  (3)  a  negative  from  the  enlarged  positive.     With 
this  negative  one  may  print  as  from  the  original  negative. 

\  390.  Lantern  Slides  from  Negatives. — In  preparing  lantern  slides  from 
photo-micrographic  or  ordinary  negatives  one  may  use  the  contact  method,  or  the 
camera.  With  the  camera  one  can  enlarge  or  reduce  to  suit  the  particular  case. 
The  camera  and  special  microscope  stand  shown  in  Fig.  181  are  admirable  for  the 
purpose.  For  lantern  slide  work  a  photographic  objective  is  used  and  the  cone 
for  enlargement  removed.  One  may  put  the  objective  in  the  front  of  the  camera 
or  in  the  middle  segment,  making  use  of  the  little  side  door. 

\  391.  Photographing  Bacterial  Cultures  in  Petri  Dishes. — For  the  successful 
photographing  of  these  cultures  dark  ground  illumination  is  employed  on  the 
principal  stated  in  §  92.  That  is  the  preparation  is  illuminated  with  rays  so 
oblique  that  none  can  enter  the  objective.  These  striking  the  culture  are  reflected 


242  PHOTO-MICROGRAPHY  \_CH.VIII 

into  the  objective.  The  clear  gelatin  around  the  growth  or  colonies  does  not 
reflect  the  light  and  therefore  the  space  between  the  colonies  is  dark. 

For  supporting  the  Petri  dishes  a  hole  is  made  in  a  front  board  for  the  camera. 
This  hole  is  slightly  larger  than  the  dish.  Over  it  is  then  screwed  or  nailed  a 
rubber  ring  slightly  smaller  than  the  Petri  dish.  This  will  stretch  and  receive  the 
dish,  and  grasp  it  firmly  so  that  it  is  in  no  danger  of  falling  out  when  put  in  a  verti- 
cal position.  If  the  camera  has  two  divisions  like  the  one  shown  the  board  with 
the  Petri  dish  is  put  in  the  front  of  the  camera,  and  the  objective  in  the  middle 
division  through  the  side  door.  Otherwise  the  board  holding  the  Petri  dish  must 
be  on  a  separate  support. 

The  illumination  is  accomplished  by  the  use  of  two  electric  lamps  with  conical 
shades.  (The  cheap  tin  shades  with  white  enamel  paint  on  the  inside  are  good). 
The  lamps  are  placed  at  the  sides  so  that  a  bright  light  is  thrown  on  the  culture, 
but  at  such  an  angle  that  none  of  it  enters  the  objective  directly. 

A  piece  of  black  velveteen  is  placed  10  to  20  cm.  beyond  the  culture.  This 
prevents  any  light  from  being  reflected  through  the  clear  gelatin  to  the  objective. 
Unless  some  such  precaution  were  taken  the  background  would  be  gray  instead  of 
black. 

One  may  use  daylight  by  putting  the  culture  in  a  support  just  outside  a  win- 
dow, leaving  the  camera  in  the  room.  The  rays  from  the  sky  are  so  oblique  that 
they  do  not  enter  the  objective.  One  must  use  a  black  non-reflecting  background 
some  distance  beyond  the  dish  as  in  using  artificial  light  (Atkinson). 

$  392.  Photographing  Bacterial  Cultures  in  Test-Tubes. — Here  the  lighting 
is  as  in  the  preceding  section,  but  a  great  difficulty  is  found  in  getting  good  re- 
sults from  the  refraction  and  reflections  of  the  curved  surfaces.  To  overcome  this 
one  applies  the  principles  discussed  in  §  144,  and  the  test-tubes  are  immersed  in  a 
bath  of  water  or  water  and  glycerin.  The  bath  must  have  plane  surfaces.  Behind 
it  is  the  black  velvet  screen,  and  the  light  is  in  front  as  for  the  Petri  dishes.  As 
suggested  by  Spitta  it  is  well  to  employ  a  bath  sufficiently  thick  in  order  that 
streak  cultures  may  be  arranged  so  that  the  sloping  surface  will  all  be  in  focus 
at  once  by  inclining  the  test-tube. 


See  the  works  on  photo-micrography  and  photography  for  the  details  of  lan- 
tern slide  making.  See  for  the  Petri  dishes  and  test-tubes,  Atkinson,  Botanical 
Gazette,  xviii  (1893),  p.  333  ;  Spitta,  Photo-Micrography  (1899),  p.  26. 


CHAPTER  IX 

CLASS    DEMONSTRATIONS    IN    HISTOLOGY    AND 
EMBRYOLOGY 


APPARATUS   AND   MATERIAL   FOR    THIS   CHAPTER 

Demonstration  microscopes,  simple  and  compound  (Figs.  195-196);  Traveling 
microscope  (Fig.  197-198);  Indicator  ocular  (Fig.  199-201);  Marker  for  putting 
rings  around  the  parts  of  specimens  to  be  demonstrated  (Fig.  61 );  Projection  mi- 
croscope (Fig.  207);  Projection  objectives  (Fig.  211-212);  Episcope  ( Fig.  214). 

DEMONSTRATION    MICROSCOPES    AND    INDICATORS 

§  393-  Simple  Microscope. — The  simple  microscope  held  in 
one  hand  and  the  specimen  in  the  other,  has  always  been  used  for 
demonstration,  but  for  class  demonstration  it  is  necessary  to  have  mi- 
croscope and  specimen  together  or  the  part  to  be  observed  by  the  class 
is  frequently  missed.  Originally  blocks  of  various  kinds  to  hold  both 
microscope  and  specimen  were  devised,  but  within  the  last  few  years 
excellent  pieces  of  apparatus  have  been  devised  by  several  opticians  for 
the  purpose.  The  accompanying  figure  shows  one  of  the  best  forms. 

FIG.  195.  Simple  Demonstra- 
tion Microscope  of  Leitz  (  Wm. 
Krafft,  N.  Y.)  As  shown  in 
the  figure  this  consists  of  a 
handle,  a  stage  and  a  lens  holder 
which  slides  up  and  down  for 
focusing.  Fot  observation  the 
student  holds  it  up  to  the  light. 

FIG.  195. 

§  394.  Compound  Demonstration  Microscope.  —This  was 
originally  called  a  clinical  or  pocket  microscope.  It  is  thus  described 
by  Mayall  in  his  Cantor  Lectures  on  the  history  of  the  microscope  :  "A 
small  microscope  was  devised  by  Tolles  for  clinical  purposes  which 
seems  to  me  so  good  in  every  way  that  I  must  ask  special  attention  for 


244 


CLASS  DEMONSTRA  T1ONS 


[  CH.  IX 


it.  The  objective  is  screwed  into  a  sliding  tube,  and  for  roughly  focus- 
ing the  sliding  motion  suffices  ;  for  fine  adjustment,  the  sheath  is  made 
to  turn  on.  a  fine  screw  thread  on  a  cylindrical  tube,  which  serves  alsa 
as  a  socket-carrier  for  the  stage.  The  compound  microscope  is  here 
reduced  to  the  simplest  form  I  have  met  with  to  be  a  really  serviceable 
instrument  for  the  purpose  in  view  ;  and  the  mechanism  is  of  thor- 
oughly substantial  character.  I  commend  this  model  to  the  notice  of 
our  opticians. ' ' 


FIG.  196.  Demonstration  compound 
microscope  of  Leitz.  Leitz  now  furnishes 
a  fine  adjustment  in  the  form  of  an  inter- 
mediate piece  between  the  objective  and 
the  tube.  This  has  in  it  a  screw  which  is 
turned  by  a  milled  ring.  For  the  object- 
ives employed  it  makes  an  efficient  fine 
adjustment  and  renders  it  possible  for  each 
person  to  adjust  the  microscope  slightly 
without  endangering  the  loss  of 'the field '. 


FIG.  196. 

Since  its  introduction  by  Tolles  many  opticians  have  produced  ex- 
cellent demonstration  microscopes  of  this  type,  but  most  of  them  have 
not  preserved  a  special  mechanism  for  fine  adjustment.  With  it  one 
can  demonstrate  with  an  objective  of  6  mm.  satisfactorily.  It  has  a  lock 
so  that  once  the  specimen  is  in  the  right  position  and  the  instrument  fo- 
cused it  may  be  passed  around  the  class.  For  observation  it  is  only 
necessary  for  each  student  to  point  the  microscope  toward  a  window  or 
a  lamp. 


CH. 


CLASS  DEMONSTRATIONS 


245 


FIG.  197. 
Traveling    microscope    set    up  for 


work     (Leitz ;    from    Wm. 


FIG.    197. 
Krafft,  N.  Y). 

A  modification  of  this  clinical  microscope  was  made  by  Zentmayer 
in  which  the  microscope  was  mounted  on  a  board  and  a  lamp  for  illum- 
inating the  object  was  placed  at  the  right  position. 


246 


CLASS  DEMONSTRATIONS 


\CH.  IX 


§  395.  Traveling  Microscope. — For  many  years  the  French 
opticians  have  produced  most  excellent  traveling  microscopes.  The 
opticians  of  other  countries  have  also  brought  out  serviceable  instru- 
ments. In  the  one  here  figured  Mr.  L,eitz  has  combined  in  an  admirable 
way  a  traveling  microscope  and  a  laboratory  instrument.  For  the 
needs  of  the  pathologist  and  sanitary  inspector  a  microscope  must  pos- 
sess compactness  and  also  the  qualities  which  render  it  usable  for  nearly 
all  the  purposes  required  in  a  laboratory.  This  instrument  is  a  type 
of  such  apparatus  which  has  grown  up  with  the  needs  of  advancing 
knowledge. 


FIG.  198. 
Krafft,  N.  Y.). 


FIG.  198. 
Traveling  microscope  folded  up  and  in  its  case  (Leitz ;  from  Wm. 


CH. 


CLASS  DEMONSTRATIONS 


247 


§  396.  Indicator  or  Pointer  Ocular. — This  is  an  ocular  in 
which  a  delicate  pointer  of  some  kind  is  placed  at  the  level  where  the 
real  image  of  the  microscope  is  produced.  It  is  placed  at  the  same 
level  as  the  ocular  micrometer,  and  the  pointer  like  the  micrometer  is 
magnified  with  the  real  image  and  appears  as  a  part  of  the  projected 
image  (§  170).  By  rotating  the  ocular  or  the  pointer  any  part  of  the 
real  image  may  be  pointed  out  as  one  uses  a  pointer  on  a  wall  or  black- 
board diagram.  By  means  of  the  indicator  eye-piece  one  can  be  cer- 
tain that  the  student  sees  the  desired  object,  and  is  not  confused  by  the 
multitude  of  other  things  present  in  the  field.  The  method  of  its  use 
is  indicated  in  Fig.  201.  This  device  has  been  invented  many  times. 
It  illustrates  well  the  adage  :  "necessity  is  the  mother  of  invention," 
for  what  teacher  has  not  been  in  despair  many  times  when  trying  to 
make  a  student  see  a  definite  object  and  neglect  the  numerous  other 
objects  in  the  field.  So  far  as  the  writer  has  been  able  to  learn, 
Quekett  was  the  first  to  introduce  an  indicator  ocular  with  a  metal 
pointer  which  was  adjustable  and  could  be  turned  to  any  part  of  the 
field  or  wholly  out  of  the  field.  See  Fig.  199,  §  126. 


FIG.  199.  FIG.  200.  FIG.  201. 

FIG.  199.  Indicator  ocular  with  metal  pointer  like  the  one  devised  by 
Quekett  (Leitz  ;  catalog}. 

FIG.  200.  Indicator  ocular  with  an  eyelash  ( cilium )  on  the  ocular  diaphragm 
to  serve  as  a  pointer  (  P) .  This  projects  about  half  way  across  the  diaphragm  open- 
ing. On  the  opposite  side  are  shown  two  rays  from  the  microscope  to  indicate  that 
the  real  image  is  formed  at  the  level  of  the  ocular  diaphragm. 

FIG.  201.  Field  of  the  microscope  with  a  mammalian  blood  preparation  to  show 
the  use  of  the  indicator  (P}  for  pointing  out  a  white  blood  corpuscle. 


248 


CLASS  DEMONSTATIONS 


[Cff.  IX 


It  is  not  known  who  adopted  the  simple  device  of  putting  the  tip 
of  a  cat's  whisker  or  an  eye-lash  on  the  diaphragm  of  the  ocular  as 
shown  in  Fig.  200.  This  may  be  done  with  any  ocular,  positive  or 
negative.  One  may  use  a  little  mucilage,  Canada  balsam  or  any 
other  cement,  and  stick  the  eyelash  on  the  upper  face  of  the  diaphragm 
so  that  it  projects  about  half  way  across  the  opening.  When  the  eye- 
lens  is  screwed  back  in  place  the  hair  should  be  in  focus.  If  it  is  not 
screw  the  eye-lens  out  a  little  and  look  again.  If  it  is  not  now  sharp,  the 
hair  is  a  little  too  high  and  should  be  depressed  a  little.  If  it  is  less 
distinct  on  screwing  out  the  ocular  it  is  too  low  and  should  be  elevated. 
One  can  soon  get  it  in  exact  focus.  Of  course  it  may  be  removed  at 
any  time. 

§  397- — Marking  the  Position  of  Objects. — In  order  that  one 
may  prepare  a  demonstration  easily  and  certainly  in  a  short  time  the 
specimens  to  be  shown  must  be  marked  in  someway.  A  very  efficient 
and  simple  method  is  to  put  rings  of  black  or  colored  shellac  around 
the  part  to  be  demonstrated.  For  this  the  Marker,  Fig.  61-62,  is 
employed  as  described  on  p.  66. 


FIG.  202. 

FIG.  202.     Ring  around  one  of  the  sections  of  a  series  for  demonstrating  some 
organ  especially  well. 


FIG.  203.  Figure  of  a  microscopical 
preparation  with  a  ring  around  a  small 
part  to  show  the  position  of  some  structural 
feature. 


CH.  IX  a]  PROJECTION  MICROSCOPE  249 


CHAPTER  IX  A 


THE  PROJECTION  MICROSCOPE 


One  of  the  most  useful  and  satisfactory  means  at  the  disposal  of 
the  teacher  of  Microscopic  Anatomy  and  Embryology  for  class  demon- 
strations is  the  Projection  Microscope.  With  it  he  can  show  two 
hundred  students  as  well  as  one,  the  objects  which  come  within  the 
range  of  the  instrument. 

It  is  far  more  satisfactory  than  microscopic  demonstrations,  for 
with  the  projection  microscope  the  teacher  can  point  out  on  the  screen 
exactly  the  structural  features  and  organs  which  he  wishes  to  demon- 
strate, and  he  can  thus  be  certain  that  the  students  know  exactly  what 
is  to  be  studied.  Unless  one  employs  a  pointer  ocular  (Fig.  201). 
there  is  no  certainty  that  the  student  selects  from  the  multitude  of 
things  in  the  microscopic  field  the  one  which  is  meant  by  the  teacher. 
Like  all  other  means,  however,  the  projection  microscope  is  limited. 
With  it  one  can  show  organs  both  adult  and  embryonic,  and  the  gen- 
eral morphology.  For  the  accurate  demonstration  of  cells  and  cell 
structure  the  microscope  itself  must  be  used.  As  a  general  statement 
concerning  the  use  of  the  projection  microscope  for  demonstration 
purposes,  it  may  be  said  that  it  is  entirely  satisfactory  for  objects  and 
details  which  show  under  the  microscope  with  objectives  up  to  16  mm. 
equivalent  focus.  For  objects  and  details  requiring  objectives  higher 
than  1 6  mm.  focus  for  ordinary  microscopic  observations,  the  projection 
microscope  is  unsatisfactory  for  large  classes. 

All  specimens  should  be  studied  under  the  microscope  by  each 
student  personally  ;  however,  it  is  much  easier  to  recognize  and 
understand  the  complex  structures  and  structural  relations  in  an  object 
after  they  have  been  demonstrated  by  the  instructor  with  the  projec- 
tion microscope. 

§  398.  Projection  Microscope. — This  is  an  arrangement  of  the 
microscope  such  that  an  image  of  the  object  under  the  microscope  is 
thrown  upon  a  screen  of  some  kind.  The  picture  on  the  screen  is 
looked  at  precisely  as  one  looks  at  the  pictures  thrown  on  the  screen 


250  PROJECTION  MICROSCOPE  \_CH.  IX  a 

by  an  ordinary  magic  lantern.  Indeed  the  projection  microscope  is  a 
magic  lantern  with  short  focus  objectives.  One  of  the  first  uses  of  the 
microscope  was  to  throw  the  images  of  various  objects  on  a  screen  so 
they  could  be  seen  by  several  persons  at  once.  The  light  used  was 
sunlight,  hence  those  early  projection  microscopes  were  called  solar  or 
sun  microscopes.  If  sunlight  were  available  at  all  times  and  could  be 
controlled,  it  would  be  universally  employed  ;  but  it  is  not  at  all  times 
available  and  whenever  it  is,  a  heliostat  is  needed  to  keep  the  light 
fixed  in  a  given  position.  Sunlight  is  therefore  practically  discarded 
and  the  electric  light  is  employed  for  illumination. 

§399.  Parts  of  a  Projection  Microscope. — These  are  shown 
in  Fig.  204  and  are  as  follows  : 

(1)  Some  form  of  projection  objective  on  a  proper  support. 

(2)  A  stage  for  supporting  the  object.     This  stage  should  be 
free  from  the  objective  holder  ;  it  should  have  a  range  of  diaphragms 
so  that  the  largest  as  well  as  the  smallest  objects  may  be  shown.     The 
stage  should  be  freely   movable  so  that  the  object  may  be  completely 
lighted  (see  Fig.  209). 

(3)  There  should  be  an  attachable  mechanical  stage  with  large 
movement  (B,  D.  1). 

(4)  A  large  lamp  condenser  for  illuminating  the  object.     If  this 
is  properly   constructed  and    the  various  parts  of   the  apparatus  are 
separately  movable  as  here  shown,  no  special  substage  condenser  is 
necessary  (see  Fig.  209). 

(5)  An  electric  arc  lamp  for  lighting. 

Fig.  204,  A,  B,  C,  D.  Half  tone  reproductions  of  photographs 
of  the  Projection  Apparatus  used  in  the  department  of  Histology  and 
Embryology,  Cornell  University.  A  10  centimeter  scale  was  photo- 
graphed with  the  apparatus  in  each  case. 

The  arc  lamp  and  the  metal  bed-piece  are  screwed  fast  to  a  thick 
board  so  that  the  whole  apparatus  may  be  moved  sidewise  or  elevated 
without  getting  any  part  out  of  line. 

From  the  weight  of  the  blocks  carrying  the  microscope,  the  stage 
and  the  condenser  and  the  width  of  the  bed-piece  no  clamping  screws 
are  necessary  for  holding  the  blocks  in  place  even  when  the  board  is 
considerably  elevated.  The  grooves  and  V's  are  so  accurately  fitted 
however  that  the  various  blocks  can  be  easily  moved  along  the  bed 
piece  and  the  centering  is  as  accurate  .in  one  position  as  in  another. 


I      i 


I 


I 


D 


E 
FIG.  205 


CH.  IX  a]  PROJECTION  MICROSCOPE  251 

A.  The  ordinary  Magic  Lantern  and  the  Projection  Microscope 
in  position  for  alternate  use  (see  Fig.  206  for  wiring,  etc.)  i,  Ammeter 
shown   at   the  left ;   2,  ordinary  stereopticon    for    lantern    slides ;    3 
Projection   Microscope.       This  shows   well  the  metal   screen   at  the 
bottom  of  the  tube,  for  cutting  off  stray  light. 

B.  Projection    Microscope   with  a  105    mm.    objective   for  very 
large  objects. 

1.  Stage  with  mechanical  stage,  large  diaphragm  opening  and 
section  of  a  cat's  brain  as  object. 

2.  105  mm.  objective  in  position  for  projecting  a  large  object  such 
as  a  brain  section. 

3.  Stage,  near  the  condenser  (See  Fig.  209). 

4.  Condenser  with  water  bath.     Under  the  condenser  is  a  focus- 
ing glass  for  photography. 

5.  90  degree,  automatic  arc  lamp. 

6.  Diaphragm   with   circular   opening   and   specimen  cooler  in 
position  (See  Fig.  208  F,  G). 

C.  Projection  Microscope  with  the  stage  and  microscope  in  posi- 
tion for  the  use  of  a  low  objective. 

i.  Large  square  diaphragm  for  the  stage.  2.  Microscope;  3, 
Stage  with  the  specimen  cooler  in  position  ;  4  Condenser  and  water 
bath  ;  5  Arc  lamp. 

D.  Projection   Microscope  with  the  parts  in  position  for  high 
powers. 

1.  Mechanical  stage  with  a  specimen  in  position. 

2.  Microscope  with  a  high  power  in  position  ;  3.  Stage  with  the 
specimen  cooler  in  position  ;  4.     Condenser  and  water  bath. 

5.     Arc  lamp  ;  6.     Large  diaphragm. 

The  white  lines  on  the  bed-piece  indicate  the  position  of  the  con- 
denser, which  is  about  8  centimeters  from  the  mica  covering  of  the 
condenser.  The  other  line  indicates  the  position  of  the  microscope  for 
high  power  projection. 

FIG.  205  A.  Zeiss'  Micro-Planar  for  projection.  (Cut  loaned  by 
Bausch  &  Lomb  Optical  Co.) 

The  micro-planars  are  of  20,  35,  50,  75,  and  100  mm.  equivalent 
focus.  When  used  for  projection  the  special  iris  in  each  should  be 
wide  open.  These  are  used  without  a  projection  ocular. 

FIG.  205.  B.  C.  Leitz  objectives  of  64  mm.  (B),  and  42  mm.  (C) 
for  micro- projection.  (Cut  loaned  by  Wm.  Krafft,  N,  Y.) 


252  PROJECTION  MICROSCOPE  \CH.   IX  a 

These  are  used  without  a  projection  ocular.  The  iris  in  each 
objective  should  be  wide  open  for  micro-projection. 

FIG.  205  D.  Projection  apparatus  showing  the  metal  hood  for  the 
lamp.  The  microscope,  the  stage  and  the  water  bath  have  been 
removed. 

A.  Screw  for  adjusting  the  upper  carbon  along  its  axis. 

B.  Wheel  by  which  the  lamp  may  be  regulated  by  hand.     It  is 
also  used  in  putting   the  carbon  holders  in  the  proper  position  for 
adding  new  carbons. 

C.  Screw  for  adjusting  the  lamp  side-wise. 

D.  Screw  for  raising  or  lowering  the  lamp. 

In  the  door  of  the  metal  covering  is  a  transparent  window  made 
of  a  piece  of  red  and  a  piece  of  blue  glass.  This  kind  of  a  window 
enables  one  to  look  at  the  arc  without  hurting  the  eyes. 

FIG.  205  B.  Half  tone  reproductions  of  the  crater  of  the  Thomp- 
son 90  degree  automatic  arc  lamp.  The  lower  carbon  shows  only  its 
tip.  In  the  center  of  the  one  at  the  left  is  shown  a  faint  shadow. 
This  is  in  the  depression  of  the  soft  core.  The  image  of  the  crater 
was  thrown  on  the  screen  by  a  64  mm.  projection  lens  (B)  and  this 
image  was  then  photographed. 

§  400.  Blackening  the  Apparatus. — The  metal  parts  of  the  en- 
tire apparatus  should  be  dead  black  to  avoid  the  reflections.  Reflec- 
tions dazzle  the  eyes  of  the  operator,  and  spreading  over  the  room, 
injure  the  brilliancy  of  the  image  on  the  screen.  The  hint  to  blacken 
the  mountings  of  the  objectives  was  given  by  Dr.  Coplin.  It  was 
found  by  personal  experience  that  not  only  the  objective  mountings 
but  every  metal  part  about  the  apparatus  should  be  blackened.  If 
necessary  one  can  do  this  himself  by  using  thin  shellac  mixed  with 
lampblack.  This  should  be  filtered  through  two  or  three  layers  of 
gauze  to  avoid  lumps.  It  can  be  put  on  with  a  soft  brush  like  a  camel's 
hair  duster.  If  the  shellac  is  too  thick  or  if  not  enough  lampblack  is 
used  the  surface  will  be  shiny.  It  should  be  a  dead  black. 

§  401.  Radiant  for  the  Projection  Microscope. — No  light  for 
projection  has  been  found  so  generally  satisfactory  as  the  electric  arc 
lamp  used  with  the  constant  current.  The  alternating  current  can  be 
used,  but  it  is  very  unsatisfactory.  For  demonstration  purposes  a  truly 
automatic  lamp  is  more  convenient  than  a  hand  feed  lamp.  In  start- 
ing the  latter  the  carbons  must  be  brought  in  contact  and  then  slightly 
separated.  With  the  automatic  lamp 'the  separation  is  attended  to  by 


CH.  IXa\ 


PROJECTION  MICROSCOPE 


253 


the  lamp  mechanism.  If  the  carbons  do  not  separate  when  an  automatic 
lamp  starts  it  is  due  to  too  small  an  amount  of  current.  If  the  current 
is  too  weak  the  magnet  is  not  strong  enough  to  lift  the  carbon. 


FIG.  206.  Diagram  showing  proper 
wiring  for  a  projection  apparatus. 

i  -f-  Main  wire  conducting  the  cur- 
rent to  the  apparatus ;  2  — ,  Main  wire 
conducting  the  current  away  from  the 
apparatus ;  j,  resistance  or  rheostat.  It 
should  be  adjustable  to  allow  a  greater  or 
less  amount  of  current  to  pass  as  occasion 
demands, 

4.  Ammeter  to  show  the  amount  of 
current.     This  is  very  desirable,  but  not 
absolutely  necessary.     If  it  is  not  used  the 
wire  would  take  the  course  indicated  by 
the  dotted  line. 

5.  Arc  lamp.       The   left  or  upper 
carbon  marked  with  the  arrow  and  plus 
sign  ( -j- )  is  the  one  in  which  is  fouud  the 
crater  giving  the  illumination  ;  6,   Arc 
lamp  similar  to  5. 

7.  Lever  of  the  double  pole,  double 
throw  switch.  The  conducting  wires  go 
to  the  binding  posts  at  the  base  of  the 
lever.  In  case  the  crater  does  not  appear 
in  the  upper  carbon,  but  in  the  one  mark- 
ed with  the  minus  sign  (  — ),  the  conduct- 
ing wires  i  and  2  are  interchanged  and 
must  be  reversed  ( §  See  406). 


%  402.  Wiring,  Rheostat  and  Ammeter  for  a  Projection  Mi- 
croscope.— Every  person  who  uses  a  projection  apparatus  should 
know  the  simple  but  fundamental  principles  involved  in  its  proper 
installation  and  running.  Fig.  206  illustrates  the  arrangement  of  all 
the  parts.  The  arrows  indicate  the  direction  of  the  current  and  the 
plus  sign  indicates  the  positive  and  the  minus  sign  the  negative 
terminal.  It  will  be  seen  that  all  the  parts,  z".  e.  the  resistance  or 
rheostat  (3)  the  ammeter  (4)  the  lamp  (5  or  6),  are  placed  in  series 
along  one  wire,  and  there  is  no  connection  with  the  other  main  wire 
(2)  until  the  current  leaves  the  lamp.  If  at  any  other  place  a  connec- 


254  PROJECTION  MICROSCOPE  [  CH.  IX  a 

tion  were  made  with  (2)  a  short  circuit  would  be  formed.  The 
absolutely  necessary  parts  in  the  installation  of  a  lantern  are  :  the 
main  wire  conveying  the  current  to  the  apparatus  (i),  the  main  wire 
conveying  the  current  from  the  apparatus  (2)  ;  some  resistance  or  a 
rheostat  (3)  somewhere  in  the  circuit  ;  and  the  ordinary  lantern  or 
the  projection  microscope  (5  or  6),  (see  A  of  Fig.  204). 

A  rheostat  of  some  form  is  absolutely  necessary.  This  part  of 
the  outfit  should  be  adjustable  and  thus  enable  one  to  use  a  current 
ranging  from  8  to  20  amperes.  If  a  fixed  rheostat  is  used  probably 
one  giving  about  12  amperes  would  be  most  generally  satisfactory. 
It  must  be  remembered  that  the  amount  of  current  at  a  given  moment 
depends  upon  the  voltage  and  also  upon  the  amount  of  current  being 
used  by  others  on  the  line.  For  really  satisfactory  projection  one 
must  have  an  adjustable  rheostat.  It  is  also_very  desirable  to  have  an 
ammeter  in  the  circuit,  then  the  current  may  be  regulated  with  some 
intelligence,  and  one  can  determine  whether  defects  in  the  illumina- 
tion are  due  to  insufficient  current  or  to  some  other  cause.  If  the 
amount  of  current  is  shown  to  be  sufficient  by  the  ammeter,  the 
cause  of  defective  projection  will  be  known  to  be  somewhere  in  the 
apparatus  and  it  can  be  hunted  down  and  corrected.  Without  an 
ammeter  one  is  tempted  to  increase  the  current  for  every  defect  in  the 
projection.  One  must  remember  also  that  the  current  is  only  one  of 
the  factors  in  successful  projection. 

While  a  rheostat  is  absolutely  necessary  one  can  omit  the  am- 
meter. If  it  is  omitted  the  main  conducting  wire  (i)  would  take  the 
course  indicated  by  the  dotted  line  just  at  the  right  of  the  ammeter 
(4).  It  should  be  said  that  the  arrangement  of  the  wires  and  the 
rheostat  and  ammeter  in  the  diagram  is  a  good  one,  but  not  the  only 
arrangement.  The  real  point  to  remember  in  installing  the  apparatus 
is  that  the  current  in  making  the  circuit  from  (i)  to  (2)  must  pass 
through  the  various  pieces  of  apparatus  in  turn,  and  not  have  it  pos- 
sible for  the  current  to  make  any  short  cuts  (short  circuits). 

§403.  Using  two  Lanterns. — (Fig.  204  A).  If  two  lanterns 
are  to  be  used  alternately  the  wiring  should  be  as  shown  in  Fig.  206. 
For  using  two  lanterns  in  this  way  the  most  convenient  switch  is  the 
double  pole  double  throw  form  (7).  The  two  main  wires  (-f-i  and 
—2)  are  connected  with  the  binding  posts  at  the  base  of  the  switch 
blades. 

Either  lamp  may  be  put  in  the  circuit  by  turning  the  lever  toward 


CH.IXa\  PROJECTION  MICROSCOPE  255 

that  lamp  until  connection  is  made.     This  arrangement  enables  one 
to  use  either  lamp  at  will. 

A  fuse  block  just  before  the  switch  is  very  desirable.  The  fuses 
should  carry  from  20  to  25  amperes  of  current. 

§404.  Arrangement  of  the  Carbons. — Originally  arc  lamps  for 
use  with  the  lantern  had  the  carbons  both  vertical.  The  "projector" 
lamps  used  at  sea,  had,  however,  the  carbons  inclined  at  an  angle  of 
30  or  40  degrees  from  the  vertical.  Lewis  Wright  (Optical  Projection, 
p.  163,)  states  that  at  his  urgent  request  a  projection  arc  lamp  for 
micro-projection  was  made  with  inclined  carbons.  Certain  it  is  that 
all  projection  lamps  have  now  the  inclined  carbons.  The  angle  of 
inclination  varies  with  different  makers.  The  lamps  furnished  by 
Zeiss  and  Reichert  with  their  apparatus  have  the  carbons  at  40  degrees 
from  the  vertical  ;  Behrens  uses  and  recommends  45  degrees.  Barnard 
and  Carver  (J.  R.  M.  S.,  1898,  p.  170)  found  by  a  careful  series  of 
experiments  that  an  angle  of  about  27  degrees  gave  the  most  satis- 
factory light. 

Within  the  last  few  years  lamps  have  been  constructed  to  hold  the 
carbons  at  right  angles,  the  horizontal  carbon  serving  for  the  illumi- 
nation. Its  brilliant  crater  faces  the  lamp  condenser,  and  it  is  believed 
that  the  illumination  with  this  arrangement  is  more  satisfactory  than 
with  inclined  carbons. 

However  the  carbons  are  arranged  in  the  lamp  success  is  attained 
only  when  the  crater  at  the  positive  pole  faces  the  condenser  and  is 
on  its  principal  axis,  and  the  negative  carbon  does  not  get  in  the  way 
and  thus  form  a  shadow.  The  proper  appearance  is  shown  in  Fig. 
205  E  of  the  90  degree  carbons,  and  in  Fig.  207  of  the  more  common 
inclined  carbons.  One  can  get  the  right  arrangement  of  the  carbons 
only  by  experience.  The  lamp  should  be  adjustable  so  that  the  posi- 
tion of  the  whole  lamp  may  be  changed  vertically  or  horizontally  to 
ensure  perfect  centering,  and  then  one  must  be  able  to  change  the 
relative  position  of  the  carbons. 

New  carbons  are  very  blunt  and  in  many  cases  need  readjusting 
after  they  have  become  sharpened  by  wear.  Their  correct  position 
and  amount  of  separation  for  a  12  to  15  ampere  current  is  shown  in 
Fig.  204. 

The  best  method  for  determining  the  character  of  the  crater  is  to 
project  the  image  of  the  two  carbons  at  the  arc  on  the  screen  with  a 
low  objective  (50  to  75  mm.  equiv.  focus).  One  can  then  mutually 


PROJECTION  MICROSCOPE 


{_CH.  IX a 


arrange  the  carbons  until  the  best  crater  is  obtained.  (See  Figs. 
205  K,  207).  One  must  always  remember  in  working  with  the  lan- 
tern or  projection  microscope  that  the  crater  should  be  in  the  upper 
carbon  (See  Fig.  207  and  §406). 

§  405.  Kind  of  Carbons  to  Employ. — The  so-called  soft  cored 
carbons  are  the  most  satisfactory.  The  crater  does  not  shift  around 
on  the  end  of  the  carbon  so  markedly  as  with  hard  carbons.  The  soft 


FIG.  207 


FIG.  207.  Front  and  side  views  of  the  carbons  of  an  arc  light  with  inclined 
carbons,  -j-  and  —  indicate  the  positive  and  negative  poles.  Compare  Figs.  204, 
206. 

A  is  a  side  view  showing  the  carbons  in  section  at  an  angle  of  30  degrees  from 
the  vertical  and  the  negative  ( — )  or  lower  carbon  slightly  in  front  of  the  positive 
( _|_)  Or  upper  carbon.  The  carbons  have  soft  cores. 

B  is  a  front  view  of  the  carbons  as  seen  projected  on  the  screen  with  a  42  mm. 
objective.  It  is  a  projection  of  the  real  image  of  the  carbons  formed  by  a  special 
achromatic  condenser  next  the  object.  This  figure  shows  that  the  source  of  light 
is  the  crater  in  the  positive  (-f)  or  upper  carbon;  it  shows  also  that  the  lower 
carbon  is  slightly  below  the  upper  carbon  as  well  as  slightly  in  front.  This  avoids 
a  shadow  from  the  lower  carbon. 

In  the  center  of  the  crater  is  shown  a  slight  shadow.  This  is  due  to  the  pit 
formed  in  the  soft  core  of  the  carbon. 


CH.IXa]  PROJECTION  MICKOSCOPE  257 

core  seems  to  furnish  a  kind  of  guide.  In  some  lamps  the  two  carbons 
are  of  equal  size  while  in  others  they  differ  in  diameter.  In  such 
cases  one  must  learn  from  the  makers  which  carbon  is  to  be  positive 
and  which  negative.  In  the  lamp  shown  in  Fig.  204,  B,  C,  D,  (A.  T. 
Thompson  &  Co.'s),  the  upper  or  positive  carbon  is  the  smaller  (i.  e. 
upper  carbon,  n  mm.;  lower  carbon  13  mm.  in  diameter). 

§  406.  How  to  Make  the  Upper  Carbon  Positive. — In  most 
cases  one  does  not  know  which  of  the  wires  in  a  circuit  is  positive  and 
which  negative.  The  simplest  way  to  proceed  is  to  make  the  connec- 
tions as  shown  in  Fig.  206  without  regard  to  which  is  positive  and 
which  negative.  When  all  is  complete  turn  on  the  current  and  allow 
it  to  pass  through  the  lamp  for  a  few  moments  and  then  open  the 
switch.  The  carbons  will  glow  for  some  time  and  the  positive  one  will 
be  much  brighter  than  the  negative  one.  If  the  upper  carbon  is  the 
bright  one  then  the  current  passes  through  the  lamp  in  the  right  direc- 
tion, but  if  the  lower  carbon  is  the  bright  one  then  the  current  passes 
through  the  lamp  in  the  wrong  direction  and  the  wires  must  be 
changed.  That  is,  in  the  diagram,  the  wire  marked  ( —  2)  is  in  place 
of  the  one  marked  (+  i).  Very  often  the  simplest  way  to  make  the 
correction  is  to  change  the  position  of  the  wires  passing  from  the  bind- 
ing posts  of  the  switch  to  the  lamp.  If  one  uses  a  plug  to  connect 
with  the  main  line  all  that  is  necessary  is  to  turn  it  the  other  side  up  if 
the  lower  carbon  is  positive,  or  if  the  plug  is  non-reversible  then  the 
wires  must  be  reversed.  It  is  well  to  mark  the  wires  in  some  way  after 
one  determines  which  is  positive  and  which  negative. 

§407.  Stage  of  the  Projection  Microscope. — This  should  be 
large  (about  10  x  12  cm.)  and  independent  of  the  rest  of  the  appa- 
ratus. It  should  be  freely  movable  toward  or  from  the  lamp  conden- 
ser so  that  objects  of  various  sizes  may  be  completely  illuminated  (see 
Fig.  209).  It  should  also  be  adjustable  vertically  so  that  it  may  be 
centered  with  the  other  pieces  of  apparatus  (Fig.  208  C.).  There 
should  be  available  an  attachable  mechanical  stage  with  a  large  range 
of  movement  both  vertically  and  horizontally  (Figs,  67 — 69,  204  B. 
and  D.).  A  specimen  cooler  in  addition  to  the  large  water  bath  is 
also  desirable  (Fig.  204  B,  C,  D,  Fig.  208  G.). 

§  408.  Openings  in  the  Stage. — For  the  projections  desirable 
in  a  modern  laboratory  of  Histology  and  Embryology  it  is  necessary 
to  be  able  to  project  objects  varying  greatly  in  size,  i.  e.  from  less 
than  i  millimeter  to  50  or  more  millimeters.  In  order  to  accomplish 


258 


PROJECTION  MICROSCOPE 


[CH.IXa 


CH.  IX a]  PROJECTION  MICROSCOPE  259 

FIG.  208  A. — H.  Diagrams  of  the  stage  and  its  parts,  its  grooved  block  and 
the  bed-piece  with  Vs.  Half  actual  size. 

A.  Large  square  diaphragm  fitting  the  still  larger  square  diaphragm  B.  ;  C. 
Vertical  stem  fitting  into  the  socket  formed  in  part  by  the  grooved  block  D.  ;  E. 
Bed-piece  with  V's  which  fit  the  grooves  of  the  blocks  carrying  the  various  parts, 
i.  e.  microscope,  stage  and  condenser  (compare  Fig.  204}.  The  bed-piece  is  like  a 
lathe  bed  and  is  of  cast  iron.  The  grooves  and  V's  are  accurately  worked  out  on  a 
machine  in  Sibley  College. 

F.  Diaphragm  with  circular  opening  ;  G.  Specimen  cooler  in  position  ;  H. 
sectional  view  showing  the  thickness  of  the  stage  and  groove,  and  V,  which  serve 
to  hold  the  diaphragms  in  position. 

this  it  is  necessary  to  have  the  stage  supplied  with  changable  dia- 
phragms to  accommodate  any  specimen  which  one  may  desire  to  pro- 
ject. This  has  been  simply  done  in  the  apparatus  shown  in  Fig.  204. 
The  thickness  of  the  stage,  the  diaphragms  and  the  methods  of  secur- 
ing the  diaphragms  are  indicated  in  Fig.  208  H.  With  this  apparatus 
(Fig.  204  B  and  Fig.  208)  objects  as  large  as  lantern  slides  may  be 
projected. 

§409.  Objective  Support  and  Focusing  Arrangement.— 
This  should  be  independent  of  the  stage  and,  for  all  but  the  lowest 
objectives,  should  have  fine  and  coarse  adjustment  as  in  the  best 
microscopes.  As  the  instrument  is  horizontal  the  fine  adjustment 
should  be  strong  enough  to  move  the  apparatus  without  the  aid  of 
gravity.  As  with  the  stage,  this  part  should  be  freely  movable  along 
the  axis  of  the  whole  apparatus  so  that  it  may  be  put  in  the  correct 
position  for  high  and  for  low  powers  and  the  coarse  and  fine  adjust- 
ment used  only  for  the  final  focusing.  It  must  also  be  adjustable 
vertically  so  that  it  may  be  accurately  centered. 

A  triple  or  quadruple  nose-piece  is  desirable  so  that  one  may  turn 
quickly  from  one  power  to  another.  Behind  the  nose-piece  should  be 
a  blackened  metal  screen  about  15  cm.  in  diameter  to  cut  off  stray 
light.  The  tube  should  be  large  and  short  so  that  it  may  not  restrict 
the  field  (2  cm.  in  diameter  and  10  cm.  in  length). 

There  should  be  a  draw-tube  with  proper  attaching  screw  so  that 
it  can  be  put  in  place  when  oculars  or  other  apparatus  needing  the 
draw-tube  are  to  be  employed. 

§  410.  Support  for  very  Low  Power  Projection. — For  pro- 
jecting objects  of  40  to  60  millimeters  in  diameter  a  photgraphic  or 
planar  objective  of  about  100  mm.  focus  answers  admirably.  This 
should  be  mounted  on  a  separate  support  and  properly  centered.  Then 


260  PROJECTION  MICROSCOPE  [_CH.   IX a 

when  it  is  to  be  used  the  ordinary  objective  support  and  focus  ar- 
rangements are  removed  bodily  and  this  apparatus  put  in  place  (Fig. 
204,  B).  It  is  focused  by  sliding  it  along  the  bed-plate. 

By  having  an  ordinary  lantern  slide  carrier  in  position  next  the 
condenser  one  may  use  this  apparatus  also  for  lantern  slide  projection 
by  removing  both  the  microscope  and  the  stage  and  putting  in  place  a 
block  carrying  the  ordinary  projection  objective.  A  double  lantern  is, 
however,  more  satisfactory  as  no  time  is  lost  in  making  the  change  of 
apparatus,  and  both  lanterns  can  be  in  perfect  adjustment  for  the  special 
purpose  in  hand. 

§411.  Water  Baths  for  Removing  Excessive  Heat. — For 
removing  the  excessive  heat  of  the  light  beam  tanks  of  water  are 
placed  in  its  path.  In  the  apparatus  shown  in  Fig.  204  the  main  tank 
is  between  the  condenser  lenses  (see  also  Fig.  209  (5)  and  Fig.  213  A). 
In  Fig.  205  D  the  water  bath  has  been  removed.  In  addition  to  this 
large  water  bath  a  special  one  called  a  specimen  cooler  is  placed  imme- 
diately under  the  stage  (Fig.  204  C,  D,  Fig.  208  G).  For  lower 
powers  where  the  specimen  is  near  the  condenser  and  hence  in  a  wide 
beam  the  large  water  bath  usually  suffices.  (See  Fig.  209,  2,  3.)  If 
the  object  is  in  or  near  the  focus  (Fig.  209  (i)),  the  heat  is  concen- 
trated as  well  as  the  light  and  the  specimen  cooler  is  necessary  to 
protect  the  specimen: 

In  some  projection  microscopes  the  cooling  tank  is  wholly  free  from 
the  condenser.  That  is  an  advantage  for  the  contact  of  the  tank  with 
the  metal  of  the  condenser  serves  to  conduct  much  heat  to  the  water 
and  it  becomes  hot  not  from  the  heat  absorbed  from  the  light,  but 
from  the  heated  metal  of  the  condenser  mounting.  To  overcome  this 
difficulty  one  may  have  two  cooling  cells  and  when  one  is  heated  a 
cold  one  can  be  put  in  its  place.  Sometimes  the  water  is  kept  cold  by 
a  constant  change  of  the  water.  This  is  liable  to  give  rise  to  currents 
and  consequently  a  shimmering  or  wavering  of  the  light  on  the  screen. 

§412.  Liquid  for  Removing  Heat. — In  the  older  works  a 
solution  of  alum  was  always  advised  as  the  heat  absorber.  It  has 
been  found  by  careful  quantitative  experiments  that  distilled  water  is 
quite  as  efficient.  It  is  much  cleaner  and  more  transparent  than  alum 
solutions.  See  Physical  Review,  vol.  i,  p.  14. 

§413.  Objectives  to  Use  in  Micro-Projection.  Without 
oculars  the  lowest  power  ordinarly  used  with  the  microscope  is  about 
75  mm.  focus.  From  this  lowest  point  one  will  find  objectives  of  64, 


CH.  IX  a}  PROJECTION  MICROSCOPE  261 

50,  42,  35,  and  25  mm.  focus  very  excellent.  Those  found  most  satis- 
factory by  the  writer  are  the  so  called  projection  objectives  to  be  used 
without  an  ocular.  For  higher  powers  than  25  mm.  the  ordinary 
objectives  used  on  the  microscope  are  mostly  employed  and,  when 
properly  selected,  are  very  satisfactory. 

For  class  demonstration,  as  stated  above,  one  can  show  to  an 
audience  of  200  with  a  screen  distance  of  5  to  8  meters  all  the  details 
which  show  satisfactorily  with  a  16  mm.  objective  and  low  ocular 
under  the  ordinary  microscope.  One  rarely  uses  with  real  satisfaction 
an  objective  higher  than  3  or  4  mm.  equivalent  focus.  One  of  from 
6  to  10  mm.  focus  is  mostly  used.  For  fine  structural  details  one 
must  use  an  ordinary  microscope.  (Bee  the  table,  §  425  for  size  of 
field  and  magnification). 

For  powers  above  3  mm.  it  is  more  satisfactory  to  have  a  screen 
distance  of  3  to  5  meters  or  even  less.  Such  demonstrations  are  only 
for  small  numbers,  and  they  must  be  near  the  screen  or  use  opera 
glasses. 

§414.  The  Use  of  Oculars  in  Micro-Projection. — The  writer 
has  found  projection  without  oculars  more  satisfactory  then  by  their 
use.  The  field  is  larger  and  higher  objectives  can  be  used,  but  the 
oculars  have  the  advantage  of  giving  increased  magnification  so  that 
one  can  get  the  same  amplification  with  lower  objectives.  A  further 
advantage  is  that  the  outlines  of  the  field  will  be  sharp  and  the  part  of 
the  specimen  shown  will  be  practically  all  in  focus.  Besides  restrict- 
ing the  field,  however,  the  oculars  reduce  the  light  greatly.  Without 
the  ocular  the  more  brilliant  image,  and  the  larger  field  are  very 
advantageous.  With  the  larger  field  the  whole  of  many  objects  may 
be  seen  and  the  relations  of  the  special  part  under  examination  noted 
at  a  glance.  It  may  be  repeated  also  that  the  micro-planars  and 
the  objectives  of  low  power  made  especially  for  projection  and  photo- 
graphy, are  designed  for  use  without  an  ocular.  In  using  these 
objectives  their  iris  diaphragm  must  be  opened  as  fully  as  possible. 

§  415.  Kind  of  Oculars. — If  an  ocular  is  used  it  may  be  either 
the  projection  oculars  like  those  for  photography  (see§  372,  A,  B),  in 
which  case  the  diaphragm  of  the  ocular  must  be  focused  on  the  screen 
to  get  the  best  results  (§  372,  B),  or  one  may  use  a  compensation  or  an 
ordinary  huygenian  ocular.  From  the  writer's  experience  an  objective 
above  8  mm.  focus  cannot  be  used  satisfactorily  for  demonstration  with 
an  ocular.  If  oculars  are  used  in  projection  it  is  better  to  have  a  screen 


262 


PROJECTION  MICROSCOPE 


[CH.IXa 


distance  not  exceeding  5  meters  ;  3  meters  would  be  even  more  satis- 
factory.    For  size  of  field  and  magnification  see  the  table  §  425. 

§  416.  Arrangement  of  the  Parts  of  the  Projection  Micro- 
scope.— As  stated  in  section  399,  all  the  parts  of  the  apparatus  should 
be  separately  adjustable.  This  is  because  the  arrangement  needs  to  be 
somewhat  different  for  different  objectives. 


FIG.  209 

FIG.  209.  To  show  the  elements  of  the  illuminating  and  condensing  apparatus 
used  in  Fig.  204,  and  the  position  in  the  cone  of  light  of  objects  of  various  sizes- 
This  figure  shows  the  necessity  of  moving  the  stage  to  accommodate  objects  of 
various  sizes  and  ensure  their  complete  illumination  by  the  entire  cone  of  light  from 
the  condenser. 

i.  Focus  of  the  condenser  where  objects  for  high  powers  are  placed.  2,  j.  Posi- 
tion of  larger  objects.  4.  Front  ten's  of  the  condenser.  5.  Laige  water  bath. 
6,  7.  Two  lenses  of  the  condenser  next  the  radiant.  8.  The  dotted  line  over  the 
meniscus  represents  the  sheet  of  mica  which  serves  to  prevent  the  too  rapid  heating 
of  the  lenses.  This  is  very  satisfactorily  held  in  position  by  a  cap  of  sheet  iron  or 
copper.  ( It  is  in  position  in  204  and  205  D. ) 

The  guiding  principle  is  that  the  specimen  should  be  lighted  by  a 
converging  cone  of  light,  and  it  should  be  lighted  by  the  entire  cone  of 
light  traversing  the  lamp  condenser.  If  one  uses  a  white  card  it  is 
easy  to  determine  the  position  and  size  of  the  cone  of  light.  If  it  is 
too  large  for  the  specimen,  either  the  lamp  condenser  is  too  near  the 
radiant  or  the  specimen  is  too  close  to  the  lamp  condenser.  (Fig.  210.) 

If  one  uses  a  substage  condenser  it  and  the  lamp  condenser  should 
be  so  placed  that  the  entire  cone  of  light  traversing  the  lamp  condenser 
can  enter  the  substage  condenser.  If  the  cone  is  too  large  they  are 
too  close  together,  or  the  lamp  condenser  is  too  near  the  radiant.  If 
the  cone  is  too  small  then  the  lamp  condenser  is  too  far  from  the 
radiant  or  the  substage  condenser,  or  perhaps  both  faults  are  present. 
One  must  remember  in  all  his  experiments  that  a  converging  cone  of 


CH.  IX  a\  PROJECTION  MICROSCOPE  263 

light  should  be  used  and  not  a  diverging  one.  The  specimen  must 
then  not  be  beyond  the  focus  of  the  lamp  condenser. 

§417.  Centering  and  Serial  Arrangement. — In  installing  a 
projection  apparatus  like  the  one  in  Fig.  204,  one  can  get  the  proper 
adjustments  first  by  making  careful  measurements.  That  is  the  upper 
carbon  should  be  opposite  the  center  of  the  lamp  condenser,  and  the 
objective  should  also  be  in  line  with  the  center  of  the  lamp  condenser. 
By  moving  the  condenser  close  to  the  lamp  and  raising  the  carbon  in 
the  position  occupied  when  in  actual  use  one  can  get  the  end  opposite 
the  axis  of  the  condenser  by  mutually  adjusting  lamp  and  condenser. 
Then  by  removing  the  stage  the  microscope  may  be  brought  close  to 
the  condenser  and  raised  or  lowered  until  the  objective  is  opposite  the 
center.  The  stage  should  then  be  put  in  position  and  adjusted  so  that 
the  center  shall  be  opposite  the  objective. 

The  purpose  of  all  this  is  to  get  all  the  pieces  centered  to  one  axis. 
If  now  the  current  is  turned  on  the  slight  adjustments  necessary  for 
the  final  centering  may  be  made  by  the  centering  screws  of  the  lamp 
(Fig.  205  D  (C  D)  ). 

The  relative  position  of  the  various  pieces  in  a  projection  micro- 
scope along  the  axis  can  be  determined  only  by  trial.  If  one  remem- 
bers that  the  object  is  to  be  illuminated  by  a  converging  cone  of  light 
it  will  aid  in  getting  the  parts  in  the  right  position.  Firstly  the  lamp 
condenser  must  be  at  the  right  distance  from  the  radiant.  Fig.  210 
shows  the  appearances  in  an  ordinary  magic  lantern  with  the  different 
relative  positions  of  radiant  and  condenser.  For  the  condenser  here 
used  (Fig.  204)  the  crater  in  the  upper  carbon  should  be  about  8  cen- 
timeters from  the  mica  sheet  over  the  condenser.  One  can  determine 
this  only  by  trial.  By  changing  the  relative  position  of  condenser  to 
radiant  and  the  object  to  the  condenser,  and  then  the  objective  and 
object  one  can  find  the  positions  giving  the  most  satisfactory  results. 
When  these  are  found  it  is  worth  while,  as  shown  in  the  picture,  to 
make  lines  on  the  fixed  part  of  the  apparatus  indicating  them.  A 
study  of  Fig.  209  will  aid  one  in  understanding  the  principles  involved 
in  the  best  relative  positions.  The  actual  cone  of  light  from  the  con- 
denser will  be  easily  seen  if  some  dust,  like  that  from  a  blackboard 
eraser  is  diffused  in  the  air  near  the  front  of  the  condenser. 

If  a  lamp  condenser  is  properly  constructed  there  is  no  necessity 
of  using  a  special  substage  condenser  if  the  different  parts  are  freely 
movable.  If,  however,  one  must  put  the  object  in  the  same  position 


264 


PROJECTION  MICROSCOPE 


[CH.  IX  a 


regardless  of  its  size  then  the  stage  must  be  nearer  the  condenser  and 
a  special  substage  condenser  used  to  bring  the  cone  of  light  from  the 
condenser  to  a  focus  more  quickly  where  one  employs  high  powers  for 
the  projection. 


,L   '''  A  3.  \^^^ 


FIG.  210 

FIG.  210.     Arrangement  and  Centering  of  the  Radiant  (Leiss}. 

In  (/)   The  Radiant,  i.  e.,  the  crater  (Fig.  209)  is  too  far  to  the  right ; 

(2)     The  crater  is  too  far  to  the  left ; 

(j )      The  crater  is  too  high  ; 

(4}     The  crater  is  too  low  ; 

(5)      1  he  crater  is  too  far  from  the  lamp  condenser  ; 

(6-7)     The  crater  is  too  near  the  condenser. 

(8)     The  crater  is  in  the  correct  position. 

As  pointed  out  in  the  explanation  of  Fig.  207,  there  may  be  a  slight  central 
shadow  with  soft  cored  carbons  when  the  lamp  and  condenser  are  in  the  best  relative 
position. 

If  one  wishes  to  make  micro-projection  a  success  it  will  be  nec- 
essary to  give  the  apparatus  the  requisite  time  and  thought.  Try  to 
understand  the  conditions  of  success  and  continue  experimenting  until 
you  have  learned  to  make  it  possible  for  the  machine  to  do  its  best  for 
you.  The  satisfaction  of  showing  a  class  real  things  is  sufficient  re- 
ward for  all  the  trouble. 

§  418.  Screen  and  Screen  Distance. — For  a  screen  nothing  is 
vSO  good  as  a  dead- white,  smooth  wall.  A  lusterless,  white  cloth 
screen  answers  well  also.  It  is  an  advantage  to  have  this  entirely 
opaque,  so  that  none  of  the  light  can  pass  through  it.  One  must 
remember  that  the  light  passing  through  the  minute  lenses  of  the 


CH.  IX  a]  PROJECTION  MICROSCOPE  265 

objective  must  be  spread  out  over  a  relatively  great  space  even  with 
low  powers,  and  over  a  much  greater  with  high  powers,  so  that  one 
cannot  afford  to  have  any  of  the  light  lost  by  transmission  through  the 
screen. 

The  distance  of  the  screen  from  the  microscope  depends  largely  on 
the  size  of  one's  audience.  The  writer  has  found  a  distance  of  five  to 
eight  meters  (26  feet)  good  for  both  low  and  high  power  projection. 
This  distance  answers  well  for  a  class  of  200  persons.  If  oculars  are 
used  with  the  objective,  a  screen  distance  of  3—5  meters  is  sufficient. 

For  the  minute  details  of  a  projected  specimen  it  is  recommended 
that  the  audience  use  opera  glasses.  These  are  also  useful  for  focus- 
ing the  image  on  the  screen. 

§419.  Darkening  the  Room. — It  is  impossible  to  succeed  in 
micro-projection  unless  the  room  can  be  made  dark,  the  darker  the 
better.  It  is  especially  important  that  the  screen  should  be  free  from 
all  light  except  that  projected  upon  it  in  forming  the  image. 

§420.  Enclosing  the  Projection  Apparatus. — It  is  desirable 
to  have  the  projection  apparatus  enclosed  as  completely  as  possible  to 
avoid  diffusing  light  through  the  room  and  thus  vitiating  the  most 
careful  darkening  of  all  windows  and  sky  lights.  It  is  also  desirable 
to  shut  in  the  light  from  the  apparatus,  as  it  dazzles  the  eyes  of  the 
operator  and  of  those  near  it  in  the  audience  so  that  the  image  on  the 
screen  cannot  be  satisfactorily  seen.  Some  forms  of  apparatus  are  en- 
closed in  a  metal  box,  others  have  a  frame  over  them  upon  which  is 
spread  black  cloth  like  silesia.  If  this  is  made  fireproof  by  soaking  it 
thoroughly  in  a  solution  of  alum,  borax  and  sodium  tungstate  it  will 
not  readily  catch  fire.  The  cloth  should  not  be  too  thick,  otherwise  it 
will  retain  too  much  heat  around  the  apparatus. 

One  should  remember  the  fundamental  law  of  vision,  viz,  that 
other  things  being  equal,  the  clearest  images  are  obtained  when  no 
light  reaches  the  eye  except  from  the  object. 

§421.  Preparations  Suitable  for  Micro-Projection.— As  a 
generalization  it  may  be  said  that  any  specimen  which  shows  clearly 
and  sharply  under  the  microscope  with  a  1 6  mm.  or  lower  objective 
will  also  give  an  excellent  projection  image.  Details  which  are  not 
visible  with  the  16  mm.  objective  are  rarely  well  brought  out  with 
sufficient  clearness  on  the  screen  for  one  or  two  hundred  people  to  see. 

(A)  The  stains  showing  best  are  those  which  are  very  transpar- 
ent, or  pure  differential  stains  like  hematoxylin.  Admirable  results 


266  PROJECTION  MICROSCOPE  \CH.  IX a 

have  been  obtained  with  hematoxylin  and  eosin,  and  the  various  car- 
mines. Every  method  of  staining  which  gives  either  sharply  differen- 
tiated results  or  transparent  colors  produces  preparations  adapted  to 
projection.  A  weak,  or  washed  out  appearance  under  the  microscope 
is  sure  to  be  even  less  satisfactory  on  the  screen. 

(B)  The  thickness  of  the  sections  may  vary  from  i/*  to  40/4.  But 
one  must  remember  that  thick  sections  are  adapted  for  low  powers 
only,  while  thin  sections,  if  well  stained,  may  be  used  with  both  high 
and  low  objectives. 

The  size  of  the  object  which  one  wishes  to  project  determines  the 
objectives  to  be  used.  By  consulting  the  table  one  can  get  a  fair  idea 
of  the  size  of  object  which  each  objective  will  satisfactorily  project. 
An  excellent  plan  to  follow  is  that  for  ordinary  microscopic  study  (see 
p.  102),  that  is,  use  first  a  low  power  to  show  the  object  as  a  whole, 
then  a  higher  one  for  details.  . 

§  422.  Projection  of  Living  Objects. — If  living  objects  are  to 
be  used  with  the  projection  microscope  it  is  nececsary  to  eliminate 
more  of  the  heat  rays  than  a  single  water  bath  can  absorb.  The  speci- 
men cooler  will  serve  for  a  short  demonstration,  but  if  one  wishes  to 
have  the  specimen  under  the  instrument  for  10  minutes  or  more  it  will 
be  necessary  to  introduce  a  second  large  water  tank  in  front  of  the  lamp 
condenser  and  to  use  the  specimen  cooler  also.  For  blood  circulation 
and  living  phenomena  generally  the  microscope  itself  is  much  more 
satisfactory  than  the  projection  microscope. 

§  423.  A  practical  suggestion  is  made  by  L,ewis  Wright  in  his  book 
on  optical  projection,  and  that  is  to  warm  the  objective  before  using  it 
for  showing  the  circulation  of  the  blood  or  in  any  case  when  a  moist 
object  is  under  it.  If  the  objective  is  cold  the  vapor  from  the  object 
will  be  condensed  on  the  objective  and  make  satisfactory  projection 
impossible. 

§  424.  Masks  for  Projection  Preparations. — The  light  used  for 
projection  is  so  brilliant  that  it  is  practically  impossible  to  arrange  the 
object  under  the  objective  with  rapidity  and  certainty  unless  there  is 
some  kind  of  guide.  The  best  one  found  so  far  is  a  mask  on  the  back 
of  the  slide  with  an  opening  for  the  preparation  to  be  shown.  This 
mask  should  be  made  of  black  paper.  One  can  cut  the  holes  in  it  with 
scissors  or  with  ticket  punches.  With  the  specimens  properly  masked, 
and  the  parts  of  the  apparatus  lustreless  black,  the  operator  can  work 
with  rapidity  and  also  with  comfort.  (Fig.  211.) 


CH.  IXa\ 


PROJECTION  MICROSCOPE 


267 


FIG.  211 

FIG.  211.  Slide  of  several  sections  with  a  black  mask,  perforated  over  the  sec- 
tions  to  be  demonstrated  with  the  projection  microscope.  This  mask  is  put  on  the 
back  of  the  slide  and  not  on  the  cover-glass. 

Unless  one  has  a  mask  something  like  this  the  light  is  so  dazzling  that  it  is 
almost  impossible  to  find  the  proper  sections.  It  is  easily  removed  by  placing  the 
slide  on  wet  blotting  paper. 


FIG.  212  A.  Leitz  Large  Micro- Projection  Apparatus  (cuts  loaned  by  Win. 
Krafft,  N.  Y.}  In  this  figure  the  apparatus  is  in  position  for  projection  with  a 
projection  ocular. 

Each  part  is.  independent  and  capable  of  special  adjustment.  (As  shown  in  the 
next  figure,  the  microscope  may  be  turned  aside,  leaving  the  apparatus  for 
ordinary  lantern  slide  projection}. 


PROJECTION  MICROSCOPE 


\_CH.IXa 


§  425.  Actual  Size  of  Objects  for  Demonstration. — In  the 
table  below  are  given  the  objectives  and  size  of  objects  which  can  be 
demonstrated  with  the  apparatus  shown  in  Fig.  204.  By  inspecting 
the  table  one  can  determine  quickly  the  size  of  a  specimen  it  is  pos- 
sible to  show  as  a  whole,  and  also  the  objective  to  employ. 

The  principles  involved  in  the  construction  of  this  table  are  those 
given  in  §  50,  §  155.  For  the  low  powers  a  micrometer  with  coarse 
lines  is  necessary.  The  diameter  of  the  screen  image  can  be  meas- 
ured off  directly,  with  a  tape  measure  or  a  measuring  rod.  It  is  of 
course  the  product  of  the  size  of  the  actual  field  and  the  magnification. 

TABLE  SHOWING    THE    OBJECTIVES,  OCULARS,  ACTUAL  SIZE 

OF  THE  FIELD,  MAGNIFICATION  AND  DIAMETER  OF 

THE  FIELD  ON  THE  SCREEN  WITH  THE 

APPARATUS  IN  FIG.  204. 


Distance  of  the  microscope  from  the  screen,  8  meters  (26  -f  feet}.  Arc  lamp 
for  illumination.  Constant  electric  current  of  no  volts  at  the  dynamo  :  12  amperes 
going  through  the  lamp. 


OBJECTIVES 

OCULARS 

ACTUAL 
DIAMETER  OF 
FIELD 

MAGNIFICA- 
TION 

DIAMETER  OF 
THE  FIELD  ON 
THE  SCREEN 

105  mm. 

None 

50.        mm. 

80    diam. 

4000    mm. 

64  mm. 

27.5 

116 

3190 

50 

12.85 

207.5 

2666+ 

42 

23- 

155 

3565 

24 

ii.  5 

326 

3749 

16 

6. 

504 

3024 

16 

Projection  X2 

1-4 

980 

1372 

16 

X2* 

3-25 

930 

3022+ 

16 

X4 

1.25 

1970 

2462+ 

8 

None 

2.625 

990 

2599— 

8 

Projection  X2 

.625 

2002 

1251 

8 

X2* 

1-5 

1940 

2910 

8 

X4 

.65 

4060 

2639 

12.5 

None 

4-25 

600 

2550 

6 

2.25 

1  120 

2520 

5 

1-5 

I600 

2400 

4 

1.5 

1680 

2520 

3 

•9 

2680 

2412 

2 

•  5375 

4OOO 

2150 

CH.  IXa\ 


PROJECTION  MICROSCOPE 


FIG.  212  B.  Leitz  large  projection  apparatus  with  the  microscope  turned  aside 
and  the  300  mm.  equivalent  focus  objective  in  place  for  projecting  a  lantern  slide. 

There  are  three  objectives  on  a  large  revolving  nose-piece.  These  serve  for 
condensers  for  different  powers  in  micro-projection,  and  when  the  microscope  is 
turned  aside,  for  projecting  lantern  slides  and  other  large  objects. 


FIG  2  f3  A.  Sectional  view  of  the  condenser 
system  and  water  bath  of  the  Bausch  &  Lomb 
Optical  CoSs  Projection  apparatus  (from  the 
Journal  of  Applied  Microscopy,  VI,  /poj). 
This  condenser  system  is  used  also  in  the  ap- 
paratus show  in  Fig.  204. 


2670 


PROJECTION  MICROSCOPE 


FIG.  213  B.      The  Micro- Projection  apparatus  of  the  Bausch  &  Lomb  Optical 
Co.     (From  the  Jour.  Ap.  Micr.  Vol.  VI.} 


FIG  213  C.  End  view  of  the  Bausch  &  Lomb  Projection  apparatus  showing 
the  means  by  which  with  the  same  instrument  either  the  microscope  or  the  ordi- 
nary projection  lens  for  lantern  slides  can  be  brought  into  position.  In  this  figure 
the  microscope  is  in  position.  (From  the  Journal  of  Applied  Microscopy, 
Vol.  VI.) 


CH.IXa} 


PROJECTION  MICROSCOPE 


26yd 


FIG  213  D.  The  Bausch  &  Loinb  Optical  Co*  s  projection  apparatus  for  objects 
in  liquids,  etc.  As  shown  the  direction  of  the  beam  from  the  lamp  is  reflected  up- 
ward by  a  45  degree  mirror  and  after  traversing  the  object  and  the  microscope  or 
ordinary  lantern  objective,  it  is  again  changed  go  degrees  by  means  of  a  prism  and 
passes  off  horizontally  to  the  screen.  From  the  prefection  of  the  reflecting  appliances 
very  little  light  is  lost.  (From  the  Jour.  Ap.  Micr.  Vol.  VI} 

§426.  How  to  demonstrate  with  the  Micro-Projection  Ap- 
paratus.— Microscopical  preparations  are  not  so  easily  used  as  are  lan- 
tern slides.  The  writer  has  found  that  the  most  successful  method  is 
for  the  teacher  himself  to  stand  by  the  apparatus,  insert  the  specimens, 
and  find  exactly  what  he  wishes  his  pupils  to  see.  Then  to  point 
them  out  a  bamboo  fish  pole  with  sharp  end  is  used.  This  should  be 
2-3  meters  long  and  if  held  out  in  the  diverging  cone  of  light  leaving 
the  microscope,  a  sharp  shadow  will  be  cast  upon  the  image.  With 
this  pointer  one  can  indicate  the  part  to  be  demonstrated  even  more 
satisfactorily  than  as  if  he  pointed  them  out  directly  on  the  screen. 

§-427.  Cleaning  the  Glass  Surfaces  of  the  Micro-projection 
Apparatus. — Inasmuch  as  it  is  so  difficult  to  make  the  light  sufficienly 
brilliant  for  micro-projection,  it  is  of  the  greatest  importance  that  all 


26ye 


PROJECTION  MICROSCOPE 


\_CH.IXa 


glass  surfaces  be  kept  as  clean  as  possible.  The  lenses  of  the  lamp 
condenser  should  be  carefully  wiped  occasionally  ;  and  the  water  bath 
should  be  opened  and  the  plane  glass  faces  thoroughly  cleaned.  It 
is  desirable  to  soak  them  in  the  cleaning  mixture  for  glass.  There  is 
always  a  certain  amount  of  deposit  on  the  glass  even  though  distilled 
water  is  used.  Every  grade  of  opacity  renders  the  image  on  the  screen 
less  excellent.  Cleanliness  is  one  of  the  most  important  requirements 
for  successful  micro-projection. 

Bach  preparation  should  be  wiped  off  before  it  is  put  in  position 
on  the  stage.  Any  particles  of  dust  are  painfully  evident  in  the  pro- 
jected image. 


FIG.  214  A 


FIG.  214  A.  The  Combined  Projection  Lantern,  Microscope  and  Mediascope  of 
Williams,  Brown  and  Earle  (cut  loaned  by  Williams,  Brown  &  Earle}. 

As  shown  in  the  figure  an  ordinary  lantern  slide  objective,  microscope  and  a 
mediascope  are  mounted  approximately  parallel  on  a  single  cast  iron  frame. 

A,  single  arc  lamp  with  a  lamp  condenser  answers  for  all.  It  may  be  moved 
opposite  the  piece  of  apparatus  to  be  used  and  thus  serve  for  givivg  the  proper  illum- 
ination. It  is  mechanically  centered  laterally  for  each  by  a  click  device. 

The  mediascope,  A.  is  a  combination  of  achromatic  lenses  of  large  aperture 
capable  of  projecting  objects  30  mm.  or  more  in  diameter.  The  objective  is  made 
adjustable  in  focus  so  that  the  screen  may  be  filled  with  the  image  of  objects  which 
vary  considerably  in  size. 


CH.  IX  a} 


PROJECTION  MICROSCOPE 


26yf 


FIG.  214 

FIG.  214.  Zeiss  Epidiascope  for  Opaque  Objects,  and  for  Transparent  Objects 
in  a  Horizontal  Position  (Zeiss'  Special  Catalog}. 

As  shown  in  this  figure  the  apparatus  is  set  up  for  opaque  objects.  For  trans- 
parent objects  M2  ( mirror  2)  is  removed  when  the  light  striking  M^  is  reflected  to 
M*  and  thence  up  through  the  object  to  M1  and  to  the  screen. 

Commencing  at  the  right :  R.  Parabolic  reflector,  which  projects  the  light  from 
the  crater  through  (  W]  the  water  bath  to  M2  the  mirror  which  is  at  the  proper  an- 
gle for  reflecting  the  light  down  upon  the  opaque  object.  From  the  opaque  object 
the  light  is  irregularly  reflected  through  the  objective  to  Ml.  M1  serves  to  reflect 
the  rays  from  the  objective  to  the  screen. 

V.  Ventilator.  M*  and  M*  are  mirrors  for  use  in  reflecting  the  light  through 
horizontal  transparent  objects. 

This  apparatus  is  designed  to  project  opaque  objects  as  large  as  22  centimeters 
in  diameter,  at  a  magnification  of  5  to  10  with  a  jo  ampere  current.  For  a  smaller 
object  one  may  magnify  as  high  as  25  diameters.  With  a  50  ampere  current  and  a 
larger  reflector  the  magnification  may  be  from  14  up  to  j/  diameters. 


PROJECTION  MICROSCOPE 


\CH.  IX a 


PROJECTION  OF  OPAQUE   OBJECTS 

$  428.  Episcope.  For  the  projection  of  opaque  objects  like  anatomical  prep- 
arations, figures  in  books,  coins  or  indeed  any  opaque  object  an  apparatus  on  the 
principle  of  the  one  figured  (Fig.  214)  is  used.  That  is,  a  powerful  light  is  thrown 
upon  the  opaque  object  and  the  rays  reflected  from  the  object  are  then  projected 
upon  the  screen  by  an  objective  as  for  a  lantern  slide.  As  the  objects  are  mostly 
in  a  horizontal  position  the  objective  points  directly  upward,  and  the  rays  from  it 
must  be  made  horizontal  by  means  of  a  45  degree  mirror  or  prism. 

This  apparatus  is  very  old.  Its  first  name  was  "aphengescope"  or  opaque  lan- 
tern. Now  it  is  called  an  episcope,  or  megascope,  and  if  for  both  opaque  and 
transparent  objects  (Fig.  214)  it  is  designated  as  an  epidiascope. 


FIG.  214  B. 

FIG.  214  B.  Spencer- Winkel  Mechanical  Stage,  New  Form.  In  this  new 
form  of  the  Spencer- Winkel  mechanical  stage  the  milled  heads  for  working  the 
screws  are  placed  close  together  so  that  they  can  be  turned  without  changing  the 


CH.  IX  a}  PROJECTION  MICROSCOPE 

position  of  the  hand.  It  is  a  very  convenient  form  of  attachable  mechanical  stage 
on  account  of  its  wide  range  of  movement  and  because  it  receives  double  slides, 
50x76  mm.  and  those  of  standard  size. 

ACKNOWLEDGEMENT 

The  Author  wishes  to  express  his  appreciation  of  help  obtained  from  Sibley 
College  in  designing  and  constructing  the  lathe  bed  and  the  movable  pieces  carry- 
ing the  parts  of  the  projection  microscope  shown  in  Fig.  204.  Thanks  are 
especially  due  to  J.  L.  Morris,  Sibley  Professor  of  Practical  Mechanics  and 
Machine  Construction  for  the  interest  shown  and  the  very  practical  help  in  direct- 
ing the  work.  Thanks  are  also  due  to  Mr.  James  Wiseman,  Foreman  in  the 
Machine  Shop  for  the  accuracy  and  excellence  with  which  the  work  was  carried 
to  completion. 

BLACKENING  TABLES 

As  stated  in  the  preceding  pages  it  is  a  great  advantage  to  have  everything 
dead  black  in  connection  with  the  Projection  Microscope.  It  is  also  desirable  to 
have  photographic  tables  and  laboratory  tables  black.  During  the  last  few  years 
an  excellent  method  of  dying  wood  with  anilin  black  has  been  devised.  This 
black  is  lustreless,  and  it  is  indestructible.  It  can  be  removed  only  by  scraping 
off  the  wood  to  a  point  deeper  than  the  stain  has  penetrated. 

It  must  be  applied  to  unwaxed  or  unvarnished  wood.  If  wax,  paint  or  var- 
nish has  been  used  on  the  tables,  that  must  be  first  removed  by  the  use  of  caustic 
potash  or  soda  or  by  scraping  or  planing.  Two  solutions  are  needed  : 

SOLUTION   A 

Copper  sulphate 125  grams 

Potassium  chlorate  or  permanganate 125  grams 

Water icoo  cc. 

Boil  these  ingredients  in  an  iron  kettle  until  they  are  dissolved.  Apply  two 
coats  of  the  hot  solution.  Let  the  first  coat  dry  before  applying  the  second. 

SOLUTION   B 

Anilin  Oil 120  cc. 

Hydrochloric  Acid 180  cc. 

Water icoo  cc. 

Mix  these  in  a  glass  vessel  putting  in  the  water  first.  Apply  two  coats  without 
heating,  but  allow  the  first  coat  to  dry  before  adding  the  second. 

When  the  second  coat  is  dry,  sand  paper  the  wood  and  dust  off  the  excess 
chemicals.  Then  wash  the  wood  well  with  water.  When  dry  sand  paper  the 
surface  and  then  rub  thoroughly  with  a  mixture  of  equal  parts  turpentine  and 
linseed  oil.  The  wood  may  appear  a  dirty  green  at  first  but  it  will  soon  become 
ebony  black.  If  the  excess  chemicals  are  not  removed  the  table  will  crock.  An 
occasional  rubbing  with  linseed  oil  and  turpentine  or  with  turpentine  alone  will 
clean  the  surface.  This  is  sometimes  called  the  Danish  method,  Denmark  black 
or  finish.  See  Jour.  Ap.  Micr.,  Vol.  I,  p.  145;  Bot.  Zeit.,  Vol.  54,  p.  326,  Bot. 
Gazette,  Vol.  24,  p.  66,  Dr.  P.  A.  Fish,  Jour.  Ap.  Micr.,  Vol.  VI.,  pp.  211-212. 


CHAPTER  X 

THE  ABBE  TEST  PLATE  AND  APERTOMETER  ;  EQUIVA- 
LENT FOCUS  OF  OBJECTIVES  AND  OCULARS  ;  DRAW- 
INGS FOR  PHOTO-ENGRAVING  ;  WAX  MODELS 


\  429.  On  the  Method  of  Using  Abbe's  Test-Plate. — This  test-plate  is  in- 
tended for  the  examination  of  objectives  with  reference  to  their  corrections  for 
spherical  and  chromatic  aberration  and  for  estimating  the  thickness  of  the  cover- 
glass  for  which  the  spherical  aberration  is  best  corrected. 

"The  test-plate  consists  of  a  series  of  cover-glasses  ranging  in  thickness  from 
0.09  mm.  to  0.24  mm.,  silvered  on  the  under  surface  and  cemented  side  by  side  on 
a  slide.  The  thickness  of  each  is  written  on  the  silver  film.  Groups  of  parallel 
lines  are  cut  through  the  film  and  these  are  so  coarsely  ruled  that  they  are  easily 
resolved  by  the  lowest  powers,  yet  from  the  extreme  thinness  of  the  silver  they 
form  a  very  delicate  test  for  objectives  of  even  the  highest  power  and  widest 
aperture.  To  examine  an  objective  of  large  aperture  the  plates  are  to  be  focused 
in  succession  observing  each  time  the  quality  of  the  image  in  the  center  of  the 
field  and  the  variation  produced  by  using  alternately  central  and  very  oblique 
illumination.  When  the  objective  is  perfectly  corrected  for  spherical  aberration 
for  the  particular  thickness  of  cover-glass  under  examination,  the  contour  of  the 
lines  in  the  center  of  the  field  will  be  perfectly  sharp  by  oblique  illumination 
without  any  nebulous  doubling  or  indistinctness  of  the  minute  irregularities  of 
the  edges.  If  after  exactly  adjusting  the  objective  for  oblique  light,  central 
illumination  is  used  no  alteration  of  the  adjustment  should  be  necessary  to  show 
the  contours  with  equal  sharpness. ' ' 

"If  an  objective  fulfills  these  conditions  with  any  one  of  the  plates  it  is  free 
from  spherical  aberration  when  used  with  cover-glasses  of  that  thickness  :  on  the 
other  hand  if  every  plate  shows  nebulous  doubling  or  an  indistinct  appearance  of 
the  edges  of  the  silver  lines,  with  oblique  illumination,  or  if  the  objective  requires 
a  different  adjustment  to  get  equal  sharpness  with  central  as  with  oblique  light, 
then  the  spherical  correction  is  more  or  less  imperfect." 

"Nebulous  doubling  with  oblique  illumination  indicates  overcorrection  of  the 
marginal  zone,  want  of  the  edges  without  marked  nebulosity  indicates  under- 
correction  of  this  zone  ;  an  alteration  of  the  adjustment  for  oblique  and  central 
illumination,  this  is,  a  difference  of  plane  between  the  image  in  the  peripheral 
and  central  portions  of  the  objective  points  to  an  absence  of  concurrent  action  of 
the  separate  zones,  which  may  be  due  to  either  an  average  under  or  overcorrection 
or  to  irregularity  in  the  convergence  of  the  rays. ' ' 

"The  test  of  chromatic  correction  is  based  on  the  character  of  the  color  bands, 
which  are  visible  by  oblique  illumination.  With  good  correction  the  edges  of  the 


CH.  X} 


TEST  PLATE  AND  APERTOMETER 


269 


silver  lines  in  the  center  of  the  field  should  show  but  narrow  color  bands  in  the 
complementary  colors  of  the  secondary  spectrum,  namely,  on  one  side  yellow- 
green  to  apple-green  on  the  other  violet  to  rose.  The  more  perfect  the  correction 
of  the  spherical  aberration  the  clearer  this  color  band  appears." 

"To  obtain  obliquity  of  illumination  extending  to  the  marginal  zone  of  the 
objective  and  a  rapid  interchange  from  oblique  to  central  light  Abbe's  illuminat- 
ing apparatus  is  very  efficient,  as  it  is  only  necessary  to  move  the  diaphragm  in 
use  nearer  to  or  further  from  the  axis  by  the  rack  and  pinion  provided  for  the 
purpose.  For  the  examination  of  immersion  objectives,  whose  aperture  as  a 
rule  is  greater  than  180°  in  air  and  those  homogeneous-immersion  objectives, 
which  considerably  exceed  this,  it  will  be  necessary  to  bring  the  under  surface  of 
the  Test-plate  into  contact  with  the  upper  lens  of  the  illluminator  by  means  of  a 
drop  of  water,  glycerin  or  oil." 

"In  this  case  the  change  from  central  to  oblique  light  may  be  easily  effected  by 
the  ordinary  concave  mirror  but  with  immersion  lenses  of  large  aperture  it  is  im- 
possible to  reach  the  marginal  zone  by  this  method,  and  the  best  effect  has  to  be 
searched  for  after  each  alteration  of  the  direction  of  the  mirror." 

"For  the  the  examination  of  objectives  of  smaller  aperture  (less  than  4o°-5o°) 
we  may  obtain  all  the  necessary  data  for  the  the  estimation  of  the  spherical  and 
chromatic  corrections  by  placing  the  concave  mirror  so  far  laterally,  that  its  edge 
is  nearly  in  the  line  of  the  optic  axis  the  incident  cone  of  rays  then  only  filling 
one-half  of  the  aperture  of  the  objective.  The  sharpness  of  the  contours  and  the 
character  of  the  color  bands  can  be  easily  estimated.  Differences  in  the  thickness 
of  the  cover-glass  within  the  ordinary  limits  are  scarcely  noticeable  with  such 
objectives." 

"It  is  of  fundamental  importance  in  employing  the  test  as  above  described  to 
have  brilliant  illumination  and  to  use  an  eye-piece  of  high  power." 

"When  from  practice  the  eye  has  learnt  to  recognize  the  finer  differences  in 
the  quality  of  the  contour  images  this  method  of  investigation  gives  very  trust- 
worthy results.  Differences  in  the  thickness  of  cover  glasses  of  o.oi  or  0.02  mm. 
can  be  recognized  with  objectives  of  2  or  3  mm.  focus." 

"With  oblique  illumination  the  light  must  always  be  thrown  perpendicularly 
to  the  direction  of  the  lines. 


FIG.  215.     The  Abbe  Test  Plate. 

"The  quality  of  the  image  outside  the  axis  is  not  dependent  on  spherical  and 
chromatic  correction  in  the  strict  sense  of  the  term.  Indistinctness  of  the  con- 
tours towards  the  borders  of  the  field  of  view  arises  as  a  rule,  from  unequal  mag- 
nification of  the  different  zones,  of  the  objective  ;  color  bands  in  the  peripheral 


270 


TEST  PLATE  AND  APERTOMETER 


\_CH.X 


portion  (with  good  color  correction  in  the  middle)  are  caused  by  unequal  magnifi- 
cation of  the  different  colored  images." 

"Imperfections  of  this  kind,  improperly  called  "curvature  of  the  field,"  are 
shown  to  a  greater  or  less  extent  in  the  best  objectives,  where  the  aperture  is  con- 
siderable." 


FIG.  216.     Abbe  Apertometer. 

\  430.  Determination  of  the  Aperture  of  Objectives  with  an  Apertometer. — 
Excellent  directions  for  using  the  Abbe  apertometer  may  be  found  in  the  Jour. 
Roy.  Micr.  Soc.,  1878,  p.  19,  and  1880,  p.  20  ;  in  Dippel,  Zimmermann  and  Czapski. 
The  following  directions  are  but  slightly  modified  from  Carpenter-Dallinger,  pp. 
394-396.  The  Abbe  apertometer  involves  the  same  principle  as  that  of  Tolles,  but 
it  is  carried  out  in  a  simpler  manner  ;  it  is  shown  in  Fig.  216.  As  seen  by  this  figure 
it  consists  of  a  semi-circular  plate  of  glass.  Along  the  straight  edge  or  chord  the 
glass  is  beveled  at  45°,  and  near  this  straight  edge  is  a  small,  perforated  circle,  the 
perforation  being  in  the  center  of  the  circle.  To  use  the  apertometer  the  micro- 
scope is  placed  in  a  vertical  position,  and  the  perforated  circle  is  put  under  the  mi- 
croscope and  accurately  focused.  The  circular  edge  of  the  apertometer  is  turned 
toward  a  window  or  plenty  of  artificial  light  so  that  the  whole  edge  is  lighted. 
When  the  objective  is  carefully  focused  on  the  perforated  circle  the  draw-tube  is 
removed  and  in  its  lower  end  is  inserted  the  special  objective  which  accompanies 
the  apertometer.  This  objective  and  the  ocular  form  a  low  power  compound  mi- 
croscope, and  with  it  the  back  lens  of  the  objective,  whose  aperature  is  to  be  meas- 
ured, is  observed.  The  draw-tube  is  inserted  and  lowered  until  the  back  lens  of 
the  objective  is  in  focus.  "In  the  image  of  the  back  lens  will  be  seen  stretched 
across,  as  it  were,  the  image  of  the  circular  part  of  the  apertometer.  It  will  ap- 
pear as  a  bright  band,  because  the  light  which  enters  normally  at  the  surf  ace  is  re- 
flected by  the  beveled  part  of  the  chord  in  a  vertical  direction  so  that  in  reality  a 
fan  of  1 80°  in  air  is  formed.  There  are  two  sliding  screens  seen  on  either  side  of 
the  apertometer  ;  they  slide  on  the  vertical  circular  portion  of  the  instrument. 
The  images  of  these  screens  can  be  seen  in  the  image  of  the  bright  band.  These 
screens  should  now  be  moved  so  that  their  edges  just  touch  the  periphery  of  the  back 
lens.  They  act,  as  it  were,  as  a  diaphragm  to  cut  the  fan  and  reduce  it,  so  that  its 
angle  just  equals  the  aperature'of  the  objective  and  no  more."  "This  angle  is 
now  determined  by  the  arc  of  glass  between  the  screens  ;  thus  we  get  an  angle  in 
glass  the  exact  equivalent  of  the  aperature  of  the  objective. '  As  the  numerical  ap- 
ertures of  these  arcs  are  engraved  on  the  apertometer  they  can  be  read  off  by  inspec- 
tion. Nevertheless  a  difficulty  is  experienced,  from  the  fact  that  it  is  not  easy  to 


CH.  X}  TEST  PLATE  AND  APERTOMETER  271 

determine  the  exact  point  at  which  the  edge  of  the  screen  touches  the  periphery 
of  the  back  lens,  or  as  we  prefer  to  designate  it,  the  limit  of  aperture,  for  curious 
as  the  expression  may  appear  we  have  found  at  times  that  the  back  lens  of  an  ob- 
jective is  larger  than  the  aperture  of  the  objective  requires.  In  that  case  the 
edges  of  the  screen  refuse  to  touch  the  periphery. ' ' 

In  determining  the  aperture  of  homogeneous  immersion  objectives  the  proper 
immersion  fluid  should  be  used  as  in  ordinary  observation.  So,  also,  with  glycerin 
or  water  immersion  objectives. 

\  431.  Testing  Homogeneous  Immersion  Liquid. — In  order  that  one  shall 
realize  the  full  benefit  of  the  homogeneous  immersion  principle  it  is  necessary 
that  the  homogeneous  immersion  liquid  shall  be  truly  homogeneous.  In  order 
that  the  ordinary  worker  may  be  able  to  test  the  liquid  used  by  him,  Professor 
Hamilton  L.  Smith  devised  a  tester  composed  of  a  slip  of  glass  in  which  was 
ground  accurately  a  small  concavity  and  another  perfectly  plain  slip  to  act  as 
cover.  (See  Proc.  Amer.  Micr.  Soc.;  1885,  p.  83).  It  will  be  readily  seen  that 
this  concavity,  if  filled  with,  air  or  any  liquid  of  less  refractive  index  than  glass, 
will  act  as  a  concave  or  dispersing  lens.  If  filled  with  a  liquid  of  greater  refractive 
index  than  glass,  the  concavity  would  act  like  a  convex  lens,  but  if  filled  with  a 
liquid  of  the  same  refractive  index  as  glass,  that  is,  liquid  optically  homogeneous 
with  glass,  then  there  would  be  no  effect  whatever. 

In  using  this  tester  the  liquid  is  placed  in  the  concavity  and  the  cover  put  on. 
This  is  best  applied  by  sliding  it  over  the  glass  with  the  concavity.  A  small 
amount  of  the  liquid  will  run  between  the  two  slips,  making  optical  contact  on 
both  surfaces.  One  should  be  careful  not  to  include  air  bubbles  in  the  concavity. 
The  surfaces  of  the  glass  are  carefully  wiped  so  that  the  image  will  not  be  ob- 
scured. An  adapter  with  society  screw  is  put  on  the  microscope  and  the  objective 
is  attached  to  its  lower  end.  In  this  adapater  a  slot  is  cut  out  of  the  right  width 
and  depth  to  receive  the  tester  which  is  just  above  the  objective.  As  object  it 
is  well  to  employ  a  stage  micrometer  and  to  measure  carefully  the  diameter  of  the 
field  without  the  tester,  then  with  the  tester  far  enough  inserted  to  permit  of  the 
passage  of  rays  through  the  glass  but  not  through  the  concavity,  and  finally  the 
concavity  is  brought  directly  over  the  back  lens  of  the  objective.  This  can  be 
easily  determined  by  removing  the  ocular  and  looking  down  the  tube. 

Following  Professor  Smith's  directions  it  is  a  good  plan  to  mark  in  some  way 
the  exact  position  of  the  tube  of  the  microscope  when  the  micrometer  is  in  focus 
without  the  tester,  then  with  the  tester  pushed  in  just  far  enough  to  allow  the  light 
to  pass  through  the  plane  glass  and  finally  when  the  light  traverses  the  concavity. 
The  size  of  the  field  should  be  noted  also  in  the  three  conditions  (\  50-52. ) 

It  will  be  seen  by  glancing  at  the  following  table  that  whenever  the  liquid  in  the 
tester  is  of  lower  index  than  glass,  that  the  concavity  with  the  liquid  acts  as  a 
concave  lens,  or  in  other  words  like  an  amplifier  (p.  109),  and  the  field  is  smaller 
than  when  no  tester  is  used.  It  will  also  be  seen  that  as  the  liquid  in  the  concav- 
ity approaches  the  glass  in  refractive  index  that  the  field  approaches  the  size 
when  no  tester  is  present.  It  is  also  plainly  shown  by  the  table  that  the  greater 
the  difference  in  refractive  index  of  the  substance  in  the  concavity  and  the  glass, 
the  more  must  the  tube  of  the  microscope  be  raised  to  restore  the  focus. 

If  a  substance  of  greater  refraction  than  glass  is  used  in  the  tester  the  field 
would  be  larger,  i.  e.,  the  magnification  less,  and  one  would  have  to  turn  the  tube 
down  instead  of  up  to  restore  the  focus. 


272 


TEST  PLATE  AND  APERTOMETER 


CH.  X-] 


The  table  given  below  indicates  the  points  with  a  tester  prepared  by  the  Gund- 
lach  Optical  Co.,  and  used  with  a  16  mm.  apochromatic  objective  of  Zeiss,  X4 
compensation  ocular,  achromatic  condenser,  i.oo  N.  A.  (Fig.  41)  : 


Tester  and  L,iquid  in  the  Concavity 

Size  of  the 
Field 

Klevation  of  the  Tube 
necessary  to 
Restore  the  Focus 

No  tester  used 

1.825  mm. 

Standard  position. 

Whole  thickness  of  the  tester  at  one  end, 
not  over  the  cavity 

.85  mm.  _ 

No  change  of  focus. 

Tester  with  water   .           _____      .     _  _     . 

.075  mm.  

Tube  raised  3^  mm. 

Tester  with  95  %  alcohol 

15  mm. 

3  mm. 

Tester  with  kerosene 

.4  mm. 

2  mm. 

Tester  with  Gundlach  Opt.  Go's  horn,  liquid 

.825  mm. 

TTfTT  mm. 

Bausch  &  L/omb  Opt.  Co.'s  horn,  liquid  
L/eitz'  horn,  liquid 

.825  mm.  
.825  mm. 

....     T2o°omm. 
T27T°7T  mm. 

Zeiss'  horn   liquid 

.825  mm. 

i2rnr  mm. 

\  432.  Equivalent  Focus  of  Objectives  and  Oculars. — To  work  out  in  proper 
mathematical  form  or  to  ascertain  experimentally  the  equivalent  foci  of  these 
complex  parts  with  real  accuracy  would  require  an  amount  of  knowledge  and  of 
apparatus  possessed  only  by  an  optician  or  a  physicist.  The  work  may  be  done, 
however,  with  sufficient  accuracy  to  supply  most  of  the  needs  of  the  working 
microscopist.  The  optical  law  on  which  the  following  is  based  is  : — "The  size  of 
object  and  image  varies  directly  as  their  distance  from  the  center  of  the  lens. ' ' 

By  referring  to  Figs.  14,  16,  21,  it  will  be  seen  that  this  law  holds  good. 
When  one  considers  compound  lens-systems  the  problem  becomes  involved,  as  the 
centre  of  the  lens  systems  is  not  easily  ascertainable  hence  it  is  not  attempted, 
and  only  an  approximately  accurate  result  is  sought. 

|  433.  Determination  of  Equivalent  Focus  of  Objectives. — Look  into  the 
upper  end  of  the  objective  and  locate  the  position  of  the  back  lens.  Indicate  the 
level  in  some  way  outside  of  the  objective.  This  is  not  the  center  of  the  object- 
ive but  serves  as  an  arbitrary  approximation.  Screw  the  objective  into  the  tube 
of  the  microscope.  If  a  Huygenian  ocular  is  used  with  the  ocular  micrometer, 
screw  off  the  field  lens  and  use  the  eye-lens  only.  If  a  positive  ocular  is  used 
no  change  need  be  made.  Pull  out  the  draw-tube  until  the  distance  between  the 
ocular  micrometer  and  the  back  lens  is  250  millimeters.  Use  a  stage  micrometer  as 
object  and  focus  carefully.  Make  the  lines  of  the  two  micrometers  parallel 
(Fig.  108).  Note  the  number  of  spaces  on  the  ocular  micrometer  required  to 
measure  one  or  more  spaces  on  the  stage  micrometer.  Suppose  the  two  microm- 
eters are  ruled  in  TV  mm.  and  that  it  required  10  spaces  on  the  ocular  micrometer 
to  enclose  2  spaces  on  the  stage  micrometer,  evidently  then  5  spaces  would  cover 
one.  The  image,  A^1  Fig  21  in  this  case  is  five  times  as  long  as  the  object,  A,B. — 
Now  if  the  size  of  object  and  image  are  directly  as  their  distance  from  the  lens  it 
follows  that  as  the  size  of  object  is  known  (-£§  mm.),  that  of  the  image  directly 
measured  (£$  mm.),  the  distance  from  the  lens  to  the  image  also  determined  in 
the  beginning,  there  remains  to  be  found  the  distance  between  the  objective  and 
the  object,  which  will  represent  approximately  the  equivalent  focus.  The  general 
formula  is,  Object,  O: Image,  I: [equivalent  focus,  F 1250.  Supplying  the  known 


CH.X]  TEST  PLATE  AND  APERTOMETER  273 


values,  O=T2ff,  !=}§  ^en  T2o  m-  :  l  mm-  :  :  F  :  25°  whence  F=5o  mm.  That  is,  the 
equivalent  focus  is  approximately  50  millimeters. 

\  434.  Determination  of  Initial  or  Independent  Magnification  of  the  Objec- 
tive. —  The  initial  magnification  means  simply  the  magnification  of  the  real  image 
(A'B1,  Fig.  21)  unaffected  by  the  ocular.  It  may  be  determined  experimentally 
exactly  as  described  in  \  433.  For  example,  the  image  of  the  object  (T2ff  mm.) 
measured  by  the  ocular  micrometer,  at  a  distance  of  250  mm.  is  yg  mm.,  i.  <?.,  it  is 
five  times  magnified,  hence  the  initial  magnification  of  the  50  mm.  objective  is 
approximately  five. 

Knowing  the  equivalent  focus  of  ah  objective,  one  can  determine  its  initial 
magnification  by  dividing  250  mm.  by  the  equivalent  focus  in  millimeters.  Thus 
the  initial  magnification  of  a  5  mm.  objective  is  -f-  —  50  ;  of  a  3  mm.,  -2-jp  =83.3  ; 
of  a  2  mm.,  *jp=  125,  etc. 

\  435.  Determining  the  Equivalent  Focus  of  an  Ocular.  —  If  one  knows  the 
initial  magnification  of  the  objective  (\  434)  the  approximate  equivalent  focus  of 
the  ocular  can  be  determined  as  follows  : 

The  field  lens  must  not  be  removed  in  this  case.  The  distance  between  the 
position  of  the  real  image,  a  position  indicated  in  the  ocular  by  a  diaphragm,  and 
the  back  lens  of  the  objective  should  be  made  250  mm.,  as  described  in  \  433,  434, 
then  by  the  aid  of  Wollaston's  camera  lucida  the  magnification  of  the  whole  mi- 
croscope is  obtained,  as  described  in  \  1  60.  As  the  initial  power  of  the  objective 
is  known,  the  power  of  the  whole  microscope  must  be  due  to  that  initial  power 
multiplied  by  the  power  of  the  ocular,  the  ocular  acting  like  a  simple  microscope 
to  magnify  the  real  image  (Fig.  21  ). 

Suppose  one  has  a  50  mm.  objective,  its  initial  power  will  be  approximately  5. 
If  with  this  objective  and  an  ocular  of  unknown  equivalent  focus  the  magnification 
of  the  whole  microscope  is  50,  then  the  real  image  or  initial  power  of  the  objective 
must  have  been  multiplied  10  fold.  Now  if  the  ocular  multiplies  the  real  image 
10  fold  it  has  the  same  multiplying  power  as  a  simple  lens  of  25  mm.  focus,  for, 
using  the  same  formula  as  before  :  0  =  5:1  =  50  ::  F:  250  whence  F  =  25.  The 
matter  as  stated  above  is  really  very  much  more  complex  than  this,  but  this  gives 
an  approximation. 

For  a  discussion  of  the  equivalent  focus  of  compound  lens-systems,  see 
modern  works  on  physics  ;  see  also  C.  R.  Cross,  on  the  Focal  Length  of  Micro- 
scopic Objectives,  Franklin  Institute  Jour.,  1870,  pp.  401-402;  Monthly  Micr. 
Jour.,  1870,  pp.  149-159  J.  J.  Woodward  on  the  Nomenclature  of  Achromatic 
Objectives,  Amer.  Jour.  Science,  1872,  pp.  406-414  ;  Monthly  Micr.  Jour.,  1872,  pp. 
66-74.  W.  S.  Franklin,  method  for  determining  focal  lengths  of  microscope 
lenses.  Physical  Review,  Vol.  f,  1893,  p.  142.  See  pp.  1119-1131  of  Carpenter- 
Dallinger  for  mathematical  formulae  ;  also  Daniell,  Physics  for  medical  students  ; 
Czapski,  Theorie  der  optischen  Instrumente  ;  Dippell,  Nageli  und  Schwendener, 
Zimmermann.  E.  M.  Nelson,  J.  R.  M.  S.  1898,  p.  362,  1900,  pp.  162-169.  Jour. 
Quekett  Micr.  Club,  vol.  V.  pp.  456,  462. 

|  436.  Drawings  for  Photo-  Engraving.  —  The  inexpensive  processes  of  repro- 
ducing drawings  bring  within  the  reach  of  every  writer  upon  scientific  subjects 
the  possibility  of  presenting  to  the  eye  by  diagrams  and  drawings  the  facts  dis- 
cussed in  the  text.  Though  artistic  ability  is  necessary  for  perfect  representation 
of  an  object,  neatness  and  care  will  enable  anyone  to  make  a  simple  illustrative  draw- 
ing, from  which  an  exact  copy  can  be  obtained  and  a  plate  prepared  for  printing. 


274  APPARA  TUS  FOR  SECTIONING  \_CH.  X 

A  careful  study  of  the  cuts  or  plates  used  to  illustrate  the  same  class  of  facts 
as  one  wishes  to  show  will  enable  one  to  produce  similar  effects.  Out- 
lines which  are  transferred  to  the  drawing  paper  may  be  obtained  by  the  camera 
lucida  or  from  a  photograph.  The  drawing  should  be  made  so  that  it  can  be 
reduced  anywhere  from  one-eighth  to  one-half.  For  ordinary  photo-engraving 
for  such  line  drawings  as  are  used  to  illustrate  this  book,  use  perfectly  black 
carbon  ink.  A  shaded  or  wash  drawing  can  be  reproduced  by  the  half-tone 
process,  also  photographs  as  is  illustrated  by  figures  190-191.  A  crayon  drawing 
on  stipple  paper  with  shadows  re-enforced  by  ink  lines  and  high  lights  scratched  out 
with  a  sharp  knife  give  admirable  results  for  anatomical  figures  by  the  half-tone 
process.  (See  for  example  the  work  of  Max  Broedel  in  Contributions  to  the  Science 
of  Medicine,  (Welch  Book)  Baltimore,  1900). 

For  photo-engravings  of  line  work  the  letters,  figures  or  words  used  to  desig- 
nate the  different  parts  can  be  put  on  the  drawing  by  pasting  letters,  etc.,  of  the 
proper  size  in  the  right  position.  In  preparing  the  block  the  photo-engraver 
eliminates  all  shadows  and  the  letters  look  as  if  printed  on  the  drawings. 

$  437.  Wax  Models. — Large  wax  models  of  the  objects  which  one  studies 
tinder  the  microscope  are  helpful  both  to  the  teacher  and  to  the  investigator. 
These  models  are  becoming  more  and  more  appreciated  for  embryologic  and 
morphologic  investigations,  for,  as  one  can  readily  appreciate,  the  effort  to  produce 
a  representation  of  the  embryo  or  organ  in  three  dimensions  helps  to  overcome 
difficulties  which  are  almost  insurmountable  if  studied  in  the  sections  alone. 

They  are  made  from  wax  plates,  the  principle  involved  being  that  the  diame- 
ter of  the  drawing  on  the  wax  plate  is  as  much  greater  than  the  object  as  the  wax 
plate  is  thicker  than  the  section. 

The  wax  plate  is  cut  with  a  sharp  instrument,  following  the  outlines  of  the 
object  which  has  been  traced  upon  it  by  the  aid  of  a  camera  lucida  or  the  projec- 
tion microscope.  The  sections  are  piled  together,  some  line  or  lines  obtained 
from  a  drawing  or  photograph  of  the  specimen  before  it  was  imbedded  and  sec- 
tioned being  used  as  a  guide  by  which  the  correct  form  of  the  pile  of  sections  can 
be  tested.  Finally  the  whole  is  welded  into  one  by  the  use  of  hot  wax  or  a  hot 
instrument.  Models  which  illustrate  complex  internal  structures  are  difficult  to 
prepare,  but  numerous  devices  will  occur  to  the  worker  as  the  representation  of 
blood  vessels  and  nerves  by  strings  or  wires.  A  large  model  will  need  much  sup- 
port which  can  be  given  by  wire  gauze,  wires,  pins  or  paper  according  to  the 
special  needs. 

A  practical  method  for  wax  modeling  was  first  published  by  G.  Born,  Arch.  f. 
Mikr.  Anat.,  Bd.  xxii,  1883,  p.  584.  The  most,  detailed  statements  of  improve- 
ments of  the  method  have  been  published  by  Born  (Bohm  u.  Oppel)  1900,  and  by 
Dr.  F.  P.  Mall  and  his  assistants.  See  contributions  to  the  Science  of  Medicine, 
pp.  926-1045.  Proceedings  of  the  Amer.  Assoc.  Anatomists,  1901,  I4th  session 
(1900)  p.  193. 

$  438.  Some  Apparatus  for  Imbedding  and  Sectioning. — As  a  supplement  to 
Chapter  VIII,  the  following  figures  of  imbedding  and  sectioning  apparatus  are 
appended.  It  will  be  noticed  that  the  microtomes  are  complex  and  consequently 
expensive.  One  is  figured  in  which  the  knife  is  moved  by  the  hands  of  the  oper- 
ator (Fig.  217).  This  form  of  instrument  is  excellent,  and  with  it  one  can  do 
all  kinds  of  work,  both  with  collodion  and  paraffin.  One  cannot  work  so  rapidly 


CH.X-] 


APPARATUS  FOR  SECTIONING 


275 


nor  with  the  same  precision.  For  much  of  the  work  one  may  section  free-hand, 
without  a  microtome.  Indeed  the  great  basis  of  histological  and  embryological 
knowledge  was  gained  by  studying  free-hand  sections  and  dissections.  At  the 
present  time  there  is  a  strong  reaction  against  the  exclusive  study  of  sections, 
and  a  tendency  to  combine  with  the  serial  sections  dissections  such  as  the  older 
anatomists  and  embryologists  made  and  gained  so  much  from. 


FIG.  217.  A  Microtome  for 
all  kinds  of  sectioning  ;  the 
knife  is  guided  by  the  top  of  the 
microtome,  but  moved  by  the 
hands  of  the  operator  (Bausch  & 
Lomb  Optical  Co. ) 


FIG.  218.  The  Minot  microtome  for  ribbon  sections  as  made  by  Bausch  and 
Lomb  Optical  Co.  It  is  arranged  for  sections  from  ///  to  25^1  and  any  intermedi- 
ate thickness. 


276 


APPARATUS  FOR  SECTIONING 


[  CH.  X 


FIG.  219.  The  Minot  microtome  for  ribbon  sections  as  made  by  the  Franklin 
Laboratory  Supply  Co.,  Boston.  This  is  to  be  made  for  2ju,  6ju,  ion,  141*,  20^1,  and, 
30)JL.  sections. 


FIG.  220. 


FIG.  221.    A. 


CH.X} 


APPARATUS  FOR  SECTIONING 


277 


FIG.  221.    B. 


FIG.  221.     C 


A  B 

FIG.    222. 

FIG.  220-222.  A  paraffin  holder  clamp  and  a  razor  support  for  the  Minot  Mi- 
crotome. (  Trans.  Amer.  Micr.  Soc.,  igoi}. 

FIG.  220.  Clamp  for  the  paraffin  block  holder.  In  A  it  is  shown  in  section,  in 
a  side  view.  With  this  clamp  one  can  use  stove  bolts  as  well  as  the  expensive  par- 
affin holders  furnished  with  the  instrument.  A  laboratory  can  have  as  many  par- 
affin block  holders  as  necessary  without  undue  expense.  ' 

FIG.  221.     Razor  Support  and  Razor. 

(A]  Support  with  heavy  base  and  vertical  piece.  The  base  should  be  capable 
of  moving  endwise  one  or  two  centimeters  to  bring  the  opening  in  the  vertical  part 
opposite  the  paraffin  block. 

(B}     Front  piece  to  the  razor  (see  Fig.  222  A ). 

( O  Razor  with  straight  back  and  edge.  By  moving  this  back  and  forth  on 
the  support  nearly  the  entire  cutting  edge  can  be  utilized. 

FIG.  222.  The  knife  support  of  the  microtome  with  the  razor  support  and 
razor  in  position. 

(A]  Front  view  ;  (B)  Back  view.  In  the  inclination  of  the  knife  toward  the 
paraffin  block  is  shown. 


278 


APPARA  TVS  FOR  SECTIONING 


\_CH.X 


FIG.  223.    Sliding  microtome -adapted  especially  for  collodion  sectioning.  (  The 
Bausch  &  Lomb  Optical  Co.). 


CH.  X] 


APPARA  TVS  FOR  SECTIONING 


279 


FIG.  224      Paraffin  dish  for  infiltrating  in  the  Lillie  oven. 
per  and  as  shown  has  a  handle  for  ease  in  transference. 
dish  in  section.     (Jour.  Appl.  Micr.  1809,  p.  266). 


It  is  made  of  cop- 
A the  whole  dish,  B  the 


FIG.  225.  The  Lillie  compartment,  paraffin  oven  for  infiltrating  tissues  with 
paraffin.  Various  sizes  of  this  are  made  ( 8,  16  and  24  compartments).  Except  for 
the  largest  laporatories  the  one  with  16  compartments  and  trays  will  be  found  of  suf- 
ficient capacity.  ( Bausch  &  Lomb  Optical  Co. ). 


280 


APPARATUS  FOR  SECTIONING 


\CH:X 


FIG.  226.  Circulation  board,  especially  for  Necturus.  This  is  prepared  from 
a  board  about  8  x  20  centimeters.  Near  one  edge  it  has  a  hole  for  a  perforated  cork. 
On  the  top  of  the  cork  is  cemented  a  thick  cover-glass  with  shellac  or  rubber  cement. 
The  cork  can  be  raised  or  lowered  in  the  board.  The  gills  of  Necturus  or  the 
web  of  a  frog"1  s  foot  can  be  spread  out  on  glass  over  the  cork.  (four.  Appl.  Micr. , 
1898,  p.  131. 


FIG.  227.  Copper  can  with  screw  top  for  collecting 
embryologic  material  and  small  aquatic  animals.  It  was 
especially  designed  for  collecting  with  a  bicycle  (Jour. 
Appl.  Micr.,  1898, p.  131). 


FIG.  228.  Egg  pipette.  This  is  made  by  putting  a 
short  piece  of  soft  rubber  tubing  over  the  end  of  a  glass 
pipette  with  rubber  bulb.  With  this  one  can  handle  the 
eggs  both  fresh  and  hardened  without  any  degree  of  in- 
jury. (Jour.  Appl.  Micr.  1898,  p. 


FIG.  227. 


FIG.  229.  Washing  apparatus  for  tissues  fixed  in  osmic  and  chromium  mix- 
tures. As  shown  in  the  figure  the  apparatus  is  connected  with  the  water  pipe  by  a 
small  side  cock.  It  is  composed  of  a  double  vessel,  the  inner  one  being  made  of  per- 
forated brass.  There  are  special  perforated -dishes  to  insert  in  the  little  compart- 
ments. This  apparatus  is  convenient  for  washing  cover-glasses,  for  the  washing 
out  for  iron  hematoxylin,  etc.  The  deeper  box  at  the  right  answers  for  the  slide 
baskets  or  holders  (Fig. 


Fig.  223a.  The  Spencer  Lens  Company's  New  Table  Micro- 
tome for  hand  sectioning.  The  vertically  moveable  socket  which 
holds  the  object  clamp  is  held  by  hardened  steel  pivot  screws  in 
two  vertically  swinging  arms  similarly  attached  to  the  main  frame, 
providing  a  movement  upon  the  parallelogram  principle,  regulated 
by  micrometer  screw  with  graduated  disc  and  index  plate.  Glass 
surface  plates  for  travelling  ways  for  the  knife. 


Ji 


Fig.  223b.  The  Spencer  Lens  Company's  New  Automatic 
Laboratory  Microtome,  with  parallelogram  principle,  giving  a 
curvilinear  knife  movement  similar  to  that  of  free  hand  section- 
ing; with  automatic  feed  and  with  independent  crank  for  quickly 
raising  or  lowering  the  object.  The  convenient  drip  pan  may  be 
quickly  unscrewed.  Furnished  when  desired  with  Gaylord's 
freezing  attachment  for  CO2  . 

The  joints  are  provided  with  hardened  steel  pivot  screws  and 
with  check  nuts.  Lubrication  not  required. 


CH.X] 


APPARATUS  FOR  SECTIONING 


281 


FIG.  229  A. — Same  as  the  preceding  with  the  inner,  perforated  box  on  edge. 


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FIG.  230.     An  inventory  card  for  the  property  in  a  department.     The  cards  are 
the  standard  sizefpr  libraries.     (Jour.  Appl.  Micr. ,  1898,  p.  124}. 


BIBLIOGRAPHY 


The  books  and  periodicals  named  below  in  alphabetical  order  pertain  wholly  or  in  part  to  the 
microscope  or  microscopical  methods.  They  are  referred  to  in  the  text  by  recognizable  abbrevi- 
ations. 

For  current  microscopical  and  histological  literature,  the  Journal  of  the  Royal  Microscopical 
Society,  the  Zoologischer  Anzeiger,  and  the  Zeitschrift  ftir  wissenschaftliche  Mikroskopie,  Ana- 
tomischer  Anzeiger,  Biologisches  Centralblatt  and  Physiologisches  Centralblatt,  the  Journal  of 
Applied  Microscopy  and  laboratory  methods  and  the  smaller  microscopical  journals  taken  to- 
gether furnish  nearly  a  complete  record. 

References  to  books  and  papers  published  in  the  past  may  be  found  in  the  periodicals  just 
named,  in  the  Index  Catalog  of  the  Surgeon  General's  library  ;  in  the  Royal  Society's  Catalog  of 
Scientific  Papers,  and  in  the  bibliographical  references  given  in  special  papers.  A  full  list  of  peri- 
odicals may  also  be  found  in  Vol.  XVI  of  the  Index  Catalog. 

BOOKS. 

Adams,  G. — Micrographia  illustrata,  or  the  microscope  explained,  etc.  Illustrated.  4th  edi- 
tion, I,ondon,  1771.  Also  Essays,  1787. 

AngstrOm. — Recherches  sur  le  spectre  solaire,  spectre  normal  du  soleil.     Upsala,  1868. 

Anthony,  Wm.  A.,  and  Brackett,  C.  F.— Elementary  text- book  of  physics.  7th  ed.  Pp.  527,  165 
Fig.  New  York,  1891. 

Barker' — Physics.     Advanced  course.     Pp.  902,  380  Fig.     New  York,  1892. 

Bausch,  E. — Manipulation  of  the  Microscope.  A  manual  for  the  work  table  and  a  text-book 
for  the  beginners  in  the  use  of  the  microscope,  illustrated.  New  Edition,  1901.  Rochester,  N.  Y. 

Beale,  I,.  S.— How  to  work  with  the  microscope.  5th  ed.  Pp.  518,  illustrated.  lyondon,  1880. 
Structure  and  methods. 

Beauregard  H.,  et  Galippe,  V. — Guide  de  1'eleve  et  du  praticien  pour  les  travaux  pratiques  de 
micographie,  comprenant  la  technique  et  les  applications  du  microscope  &  1'histologie  vegetale, 
h,  la  physiologic,  a.  la  clinique,  &  la  hygiene  et  &  la  medicine  legale.  Pp.  904,  570  Fig.  Paris,  1880. 

Behrens,  H — Mikrochemische  Technik,  2d  ed.     Pp.  68.     Hamburg,  1900. 

Behrens,  T.  H. — Anleitung  zur  microchemischen  Analyse  der  wichtigsten  organischen  Ver- 
bindungen.  Hamburg,  1895-1897. 

Behrens,  T.  H. — Transl,  by  J.  W.  Judd.  A  manual  of  microchemical  analysis  with  an  intro- 
ductory chapter  by  J.  W.  Judd.  I,ondon,  1894. 

Behrens,  W.— The  microscope  in  botany.  A  guide  for  the  microscopical  investigation  of 
vegetable  substances.  Translated  and  edited  by  Hervey  and  Ward.  Pp.  466,  illustrated.  Boston, 
1885. 

Behrens,  W.— Tabellen  zum  Gebrauch,  bei  mikroskopischen  Arbeiten.'  3d  edition.  Pp.  237. 
Braunschweig,  1898. 

Behrens,  W.,  Kossel,  A.,  und  Schiefferdecker,  P.— Das  Mikroscop  und  die  Methoden  der 
mikroskopischen  Uuntersuchung.  Pp.  315,  193  Fig.  Bravtnschweig,  1889  +. 

Boehm,  A.  A.  und  von  Davidoff,  M. — A  text-book  of  Histology,  including  microscopic  tech- 
nique, from  the  2d  German  edition.  Translated  by  H.  H.  Gushing  and  edited  by  G.  C.  Huber. 
Pp.  501,  illustrated.  Philadelphia  and  I,ondon,  1900. 

-     Boehm,  A.  und  Oppel,  A. — Taschenbuch  der  mikroskopischen  Technik,  4th  ed.  with  direc- 
tions by  Born  for  making  wax  models.     Pp.  240.     Mtinchen,  1900. 

Brewster,  Sir  David. — A  treatise  on  the  mikroscope.  From  the  7th  ed.  of  the  Encyc.  Brit., 
with  additions.  Illustrated,  1837. 

Brewster,  .Sir  David. — A  treatise  on  optics.     Illustrated.     New  edition.     I,ondon,  1853. 

Browning,  J. — How  to  work  with  the  micro-spectroscope.     L,ondon,  1894. 


BIBLIOGRAPHY  283 

Bousfield,  E.  C. — Guide  to  photo-micrography.     2d  ed.     Illustrated.     London,  1892. 

Carnoy,  J.  B.  L,e  Chanoine. — La  Biologic  Cellulaire  ;  Etude  comparee  de  la  cellule  dans  les 
deux  regnes.  Illustrated  (incomplete).  Paris,  1884.  Structure  and  methods. 

Carpenter,  W.  B. — The  microscope  and  its  revelations.  6th  ed.  Pp.  882,  illustrated.  London, 
and  Philadelphia,  1881.  Methods  and  structure. 

Carpenter- Dallinger. — The  microscope  and  its  revelations,  by  the  the  late  William  B.  Carpen- 
ter. 8th  edition,  in  which  the  ist  seven  and  the  23d  chapters  have  been  entirely  re-written,  and 
the  text  throughout  reconstructed,  enlaiged  and  revised  by  the  Rev.  W.  H.  Dallinger.  22  plates 
and  nearly  900  wood  engravings.  Pp.  1181.  London  and  Philadelphia,  1901. 

Chamberlain,  C.  J. — Methods  in  plant  histology,  Illustrated.     Chicago,  1901. 

Clark,  C.  H. — Practical  methods  in  microscopy.     Illustrated.     Boston,  1894. 

Cooke,  M.  C. — One  thousand  objects  for  the  microscope.  Pp.  123.  London,  no  date.  500  fig- 
ures and  brief  descriptions  of  pretty  objects  for  the  microscope. 

Crookshank,  E.  M. — Photography  of  bacteria.    London  and  New  York,  1887. 

Cross  and  Cole.— Modern  microscopy  for  beginners.  Part  I.  The  microscope  and  instruc- 
tions for  its  use.  Part  II.  Microscopic  objects,  how  prepared  and  mounted.  Illustrated.  2d 
edition,  London,  1895. 

Czapski,  Dr.  Siegfried. — Theorie  der  optischen  Instrumente  nach  Abbe.  Illustrated.  Bres- 
lau,  1893. 

Dana,  J.  D. — A  system  of  mineralogy.     Illustrated.    6th  ed.     New  York,  1892. 

Daniell,  A. — A  text-book  of  the  principles  of  physics.     Illustrated.     3d  ed.  London,  1895. 

Daniell,  A. — Physics  for  students  of  medicine.     Illustrated.     London  and  New  York,  1896. 

Davis,  G. — Practical  microscopy.    3d  ed.     Illustrated.    London,  1895. 

Dippel,  L. — Das  Mikroskop  und  seine  Anwendung.     Illustrated.     Braunschweig,  1898. 

Dodge,  Charles  Wright. — Introduction  to  elementary  practical  biology  ;  a  laboratory  guide  for 
high  school  and  college  students.  New  York,  1894. 

Eberth,  C.  J. — Friedlander's  mikroskopische  Techtiik  zum  gebiauche  bei  mediciiiischen  und 
pathologische-anatomischen  Untersuchungen.  Pp.  359,  illustrated.  Berlin,  1900. 

Ebner,  V.  V. — Untersuchungen  tiber  die  Ursachen  der  Anisotropie  organischer  Substanzen. 
Leipzig,  1882.  Large  number  of  references. 

Ellenberger,  W. — Handbuch  der  vergleichenden  Histologie  und  Physiologic  der  Haussauge- 
thiere.  2d  edition.  Berlin,  1901 -r. 

Fol,  H. — Lehrbuch  der  vergleichenden  mikroskopischen  Anatomic,  mit  Einschluss  der  ver- 
gleichenden Histologie  und  Histogenie.  Illustrated  (incomplete).  Leipzig,  1884.  Methods  and 
structure. 

Foster,  Frank  P. — An  illustrated  encyclopaedic  medical  dictionary,  being  a  dictionary  of  the 
technical  terms  used  by  writers  on  medicine  and  the  collateral  sciences  in  the  Latin,  English, 
French  and  German  languages.  Illustrated,  four  quarto  volumes.  1888-1893. 

Fraenkel  und  Pfeiffer.— Atlas  der  Bacterien-Kunde.     Berlin,  1889+. 

Francotte,  P. — Manuel  de  technique  microscopique.     Pp.  433,  no  Fig.     Brussels,  1886. 

Francotte,  P.— Microphotographie  appliquee  a  Thistologie,  1'anatomie  comparee  et  1'embryolo- 
gie.  Brussels,  1886. 

Frey,  H.— The  microscope  and  microscopical  technology.  Translated  and  edited  by  G.  R. 
Cutter.  Pp.  624,  illustrated.  New  York,  1880.  Methods  and  structure. 

Gait,  H. — The  microscopy  of  the  starches  illustrated  by  photomicrographs.     London,  1900. 

Gamgee,  A. — A  text-book  of  the  physiological  chemistry  o.f  the  animal  body.  Part  I,  pp.  487, 
63  Fig.  London  and  New  York,  1880.  Part,  II,  1893. 

Gebhardt,  W. — Die  mikrophotographische  Aufnahme  gefarbter  Praparate.  Illustrated. 
Miinchen,  1899. 

Giltay,  Dr.  E.— Sieben  Objecte  unter  dem  Mikroskop.  Einf uhrung  in  die  Grundlehren  der 
Mikroskopie.  Leiden,  1893.  This  is  also  published  in  the  Holland  (Dutch)  and  French  language. 

Goodale,  G.  L-  Physiological  botany.  Pp.  499  +  36,  illustrated.  New  York,  1885.  Structure 
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Gould,  G.  M. — The  illustrated  dictionary  of  medicine,  biology  and  allied  sciences.  Illustrated, 
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especially  satisfactory  for  the  worker  with  the  microscope. 

Hager,  H.  und  Mez.  C. — Das  mikroskop  und  Seine  Anwendung.  8th  ed.,  revised  and  en- 
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Halliburton,  W.  D. — A  text-book  of  chemical  physiology  and  pathology.  Pp.  874,  104  illus- 
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Hanausek,  T.  F. — L,ehrbuch  der  mikroskopischen  Technik.     Illustrated.     Stuttgart,  1900. 

Harker,  H. — Petrology  for  students  ;  an  introduction  to  the  study  of  rocks  under  the  micro- 
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Herzfeld,  T,  J. — The  technical  testing  of  yarns  and  textile  fabrics  with  reference  to  official 
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Hogg,  J.— The  microscope,  its  histoiy,  construction  and  application,  isth  edition,  illustrated. 
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L,ehmann,  O. — Molekularphysik  mit  besonderer  Berilcksichtigung  mikroskopischer  Unter- 
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Mace,  E. — I,es  substances  alimentaire  etudies  au  microscope  surtout  au  point  de  vue  de  leurs 
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Wood,  J.  G. — Common  objects  for  the  microscope.  Pp.  132.  London,  no  date.  Upwards  of 
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Wormly,  T.  G. — The  micro-chemistry  of  poisons.     2d  ed.     Pp.  742,  illus.     Philadelphia,  1885. 

Wright,  Lewis. — Optical  Projection,  a  treatise  on  the  use  of  the  lantern  in  exhibition  and 
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See  also  Watt's  chemical  dictionary,  and  the  various  general  and  technical  encyclopedias. 

PERIODICALS* 

The  American  journal  of  anatomy  (including  histology,  embryology  and  cytology).  Balti- 
more, 1901  +  . 

The  American  journal  of  medical  research,  Boston,  1901  +  . 

The  American  Journal  of  physiology.     Boston,  1896+. 

The  American  journal  of  microscopy  and  popular  science.     Illustrated.     New  York,  1876-1881. 

The  American  monthly  microscopical  journal.     Illustrated.     Washington,  D.  C.,  1880  + . 

American  naturalist.     Illustrated.     Salem  and  Philadelphia,  1867-!-. 

American  quarterly  microscopical  journal,  containing  the  transactions  of  the  New  York 
microscopical  society.  Illustrated.  New  York,  1878-4-. 

American  microscopical  society,  Proceedings.     1878-1894 ;  Transactions,  1895+. 


*NOTE— When  a  periodical  is  no  longer  published,  the  dates  of  the  first  and  last  volumes  are 
given  ;  but  if  still  being  published,  the  date  of  the  first  volume  is  followed  by  a  plus  sign. 

See  Vol.  XVI  of  the  index  Catalog  of  the  Library  of  the  Surgeon  General's  office  for  a  full 
list  of  periodicals. 


288  BIBLIOGRAPHY 

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1886+.  Besides  articles  relating  to  the  microscope  or  histology,  a  full  record  of  current  anatomi- 
cal literature  is  given. 

Annales  de  la  societe  beige  de  microscopic.     Bruxelles,  1874  f . 

Archives  d'Anatomie  microscopique.     Illustrated.     Paris,  1897.     (Balbiani  et  Ranvier). 

Archiv  ftir  miroscopische  Anatomic.     Illustrated.     Bonn,  1865+. 

Centrablatt  ftir  Physiologic.  Unter  Mitwirkung  der  physiologischen  Gesellschaft  zu  Berlin 
Herausgegeben  von  S.  Exner  und  J.  Gad.  Leipzig  and  Wien.  1887  +  .  Brief  extracts  of  papers 
having  a  physiological  bearing.  Full  bibliography  of  current  literature. 

English  mechanic.  L,ondon,  1866+ .  Contains  many  of  the  papers  of  Mr.  Nelson  on  light- 
ing, photo-micrography,  etc. 

Index  medicus.     New  York,  1879+.     Bibliography,  including  histology  and  microscopy. 

International  journal  of  micioscopy  and  popular  science.     L,otidon,  1890+. 

Journal  of  anatomy  and  physiology.     Illustrated.     L,ondon  and  Cambridge,  1867+. 

Journal  of  Applied  Microscopy  and  Laboratory  methods.  Illustrated.  Rochester,  N.  Y., 
1898+ . 

Journal  de  micrographie.     Illustrated.     Paris,  1877-1892. 

Journal  of  microscopy  and  natural  science.    L,ondon,  1885  +  . 

Journal  of  the  New  York  microscopical  society.     Illustrated.     New  York,  1885+. 

Journal  of  physiology.     Illustrated.     L,ondon  and  Cambridge,  1878+. 

Journal  of  the  American  chemical  society.     New  York,  1879+. 

Journal  of  the  chemical  society.     L/ondon,  1848  +  . 

Journal  of  the  royal  microscopical  society.  Illustrated.  L,ondon,  1878+.  Bibliography  of 
works  and  papers  relating  to  the  microscope,  microscopical  methods  and  histology.  It  also  in- 
cludes a  summary  of  many  of  the  papers. 

Journal  of  the  Quekett  microscopical  club.     L/ondon,  1868 +  . 

The  L,ens,  a  quarterly  journal  of  microscopy,  and  the  allied  natural  sciences,  with  the  tran- 
sactions of  the  state  microscopical  society  of  Illinois.  Chicago,  1872-1873. 

The  Metallographist,  a  quarterly  publication  devoted  to  the  study  of  metals  with  special 
reference  to  their  physics,  microstructure,  their  industrial  treatment  and  application.  Illustrated 
especially  by  photo-micrographs  of  metals  and  alloys.  Boston,  1898+. 

The  Microscope.     Illustrated.    Washington,  D.  C.,  1881-1897. 

Microscopical  bulletin  and  science  news.  Illustrated.  Philadelphia,  1883  +  .  The  editor,  Ed- 
ward Pennock  introduced  the  term  "par-focal  "  for  oculars  (see  vol.  iii,  p  31). 

Monthly  microscopical  journal.     Illustrated.     L,ondon,  1869-1877. 

Nature.     Illustrated.    London,  1869+. 

The  Observer.     Portland,  Conn.,  1890-1897. 

Philosophical  Transactions  of  the  Royal  Society  of  L,ondon.     Illustrated.     L,ondon,  1665+. 

Proceedings  of  the  American  microscopical  society,  1878+. 

Proceedings  of  the  Royal  Society.    London,  1854+. 

Quarterly  journal  of  microscopical  science.     Illustrated.    L,ondon,  1853  + . 

Science  Record.    Boston,  1883-4. 

Zeitschrift  f.  Angewandte.     Mikroskopie.     1898+. 

Zeitschrift  f tir  Instrumentenkunde.     Berlin,  1881+. 

Zeitschrift  ftlr  physiologische  Chemie.     Strassburg,  1877+. 

Zeitschrift  ftir  wissenschaftliche  Mikroskpie  und  ftlr  mikroskopische  Technik.  Illustrated. 
Braunschweig.  1884  + .  Methods,  bibliography  and  original  papers. 

Besides  the  above-named  periodicals,  articles  on  the  microscope  or  the  application  of  the 
microscope  appear  occasionally  in  nearly  all  of  the  scientific  journals.  One  is  likely  to  get 
references  to  these  articles  through  the  Jour.  Roy.  Micr.  Soc.  or  the  Zeit.  wiss.  Mikroskopie. 
Excellent  articles  on  Photo-micrography  occur  in  the  special  Journals  and  Annuals  of  Photog- 
raphy. 


BIBLIOGRAPHY  288  a 

ADDITIONAL   BIBLIOGRAPHY 

Bagshaw,  W. — Elementary  Photomicrography.    Illustrated.     Pp.  70.    I^ondon,  1902. 

Bausch,  Edward. — Use  and  care  of  the  Microscope  (Extracts  from  Manipulation  of  the  Micro- 
scope). Rochester,  1902.  This  booklet  should  be  in  the  hands  of  every  beginner  in  microscopy. 
It  is  furnished  free  to  laboratory  teachers. 

Beck  and  Andrews. — Photographic  lenses.  I^ondon,  1903.  Many  illustrations  showing  vari- 
ous forms  of  photographic  objectives  and  the  work  for  which  they  are  adapted. 

Cabot,  R.  C. — A  guide  to  the  clinical  examination  of  the  blood  for  diagnostic  purposes.  3d 
edition,  Illustrated.  New  York,  1903. 

Encyclopadie  der  Mikroskopischen  Technik.  Herausgegeben  von  :  Ehrlich,  Krause,  Mosse, 
Rosin  und  Weigert.  Pp.  1400,  Berlin  and  Vienna,  1903. 

Ewing,  James.— Clinical  Pathology  of  the  Blood.    2d  Edition.   Illustrated.    Philadelphia,  1903; 

Gay  lord  and  Aschoff.— The  Principles  of  Pathological  Histology,  Philadelphia,  1901.  The 
illustrations  are  mostly  photo-micrographs.  Part  I  gives  a  resume  of  microscopical  technique. 
Part  III  is  on  the  principles  of  optics  and  photomicrography. 

Greenish,  H  G. — Microscopical  examination  of  food  and  drugs.  Illustrated.  Pp.  24  and  321. 
I,ondon  and  Philadelphia,  1903. 

Hardesty,  Irving. — Neurological  Technique.  Pp.  183.  Chicago  and  I^ondon,  1902.  labora- 
tory directions  for  the  dissection  of  the  central  nervous  system  and  Bna  neurological  nomen- 
clature. 

Havestadt,  Dr.  H.— Translated  by  J.  D.  and  Alice  Everett.  Jena  Glass.  Illustrated.  Pp.  xiv 
and  419.  L,ondon  and  New  York,  1903. 

Heisler,  J.  C. — A  text-book  of  Embryology,  for  students  of  medicine.  2d  revised  edition. 
Pp.  405.  Illustrated.  Philadelphia,  1902. 

Hiorns.  Arthur  H.— Metallography,  an  introduction  to  the  study  of  the  structure  of  metals, 
chiefly  by  the  aid  of  the  microscope.  Pp.  158.  Illustrated.  I,ondon,  1902. 

von  HOhnel  F.  R.— Die  Mikroskopie  der  technisch-verwandten  Fasserstoffe.    Vienna,  1887. 

Kaiserling,  C. — I<ehrbuch  der  Mikrophotographie  nebst  Bemerkungen  tlber  VergrOsserungen 
und  Projection.  Illustrated.  Pp.  179.  Berlin,  1903. 

Koristka,  Ditta  F. — Di  Microscopi  ed  accessor!.    Milano,  1902. 

L,angley,  J.  N.— Practical  Histology.    Illustrated.     Pp.  340.    lyOndon  and  New  York,  1901. 

L,econte,  H. — I^es  textiles  vegetaux,  leur  examen  microchimique.     Paris. 

L,indner,  P. — Atlas  der  mikroskopischen  Grundlagen  der  Gahrungskunde.  Illustrated. 
Berlin,  1903. 

Mallory,  F.  B.,  and  Wright,  J.  H.— Pathological  Technique.  A  practical  manual  for  workers 
in  pathological  histology  and  bacteriology  including  directions  for  the  performance  of  autopsies 
and  for  clinical  diagnosis  by  laboratory  methods.  2d  revised  edition.  Pp.  432.  Illustrated. 
Philadelphia,  1901. 

Mann,  Gustav. — Physiological  Histology.     Methods  and  Theory.     Pp.  488.     Oxford,  1902. 

McMurrich,  J.  P.— The  development  of  the  Human  Body.  A  manual  of  human  embryology. 
Illustrated.  Pp.  527.  Philadelphia,  1902. 

Mell,  P.  H.— Biological  laboratory  Methods.  Illustrated.  Pp.  321.  New  York  and  Condon, 
1902. 

Minot,  Charles  S.— A  laboratory  text-book  of  Embryology.  Illustrated.  Pp.  380.  Phila- 
delphia, 1903.  Full  and  satisfactory  methods  for  Embryological  work. 

Molisch,  H. — Grundriss  einer  Histochemie  der  pflanzlichen  Genussmittel.    Jena  1891. 

Moore,  V.  A. — laboratory  Directions  for  beginners  in  Bacteriology.  Illustrated.  2d  edition. 
Boston,  1900. 

Nelson,  Edward  M. — A  bibliography  of  works  (dated  not  later  than  1700)  dealing  with  the 
microscope  and  other  optical  subjects.  Journal  of  the  Royal  Microscopical  Society  for  the  Year 
1902,  pp.  20-23. 


288  b  BIBLIOGRAPHY 

Oertel.  T.  E.— Medical  Microscopy.     Illustrated.     Philadelphia,  1902, 

Williams,  H.  U.— Bacteriology.    4th  edition.    Illustrated.     Philadelphia,  1903. 

Piersol,  G.  A.— Text-book  of  Normal  Histology  including  an  account  of  the  development  of 
the  tissues  and  of  the  organs.  Also  technique.  Illustrated.  Pp.  439.  Philadelphia,  1893-1899. 

Pozzi-Escot. — Analyse  microchimique.     Paris. 

Reference  Handbook  of  the  Medical  Sciences.  8.  vols.  4°.  New  edition  completely  revised 
and  rewritten.  New  York,  1900-1905. 

Schaefer,  E.  A.— The  essentials  of  Histology,  descriptive  and  practical,  for  the  use  of  students. 
6th  edition,  revised  and  enlarged.  Illustrated.  Pp.  416.  Methods  are  given  also. 

Szymonowicz,  I,,  translated  by  MacCallum,  J.  B.— A  text-book  of  Histology  and  Microscopic 
Anatomy  of  the  human  body,  including  microscopic  technique.  Illustrated.  Pp.  435.  Phila- 
delphia and  New  York,  1902. 

Walmsley,  W.  H.— The  A,  B,  C  of  Photo-Micrography.  A  practical  handbook  for  beginners. 
Plates  and  text  figures.  New  York,  1902. 

PERIODICALS 

The  American  Journal  of  Anatomy,  Baltimore,  1901  +.  The  American  Journal  of  Anatomy 
including  Histology,  Embryology  and  Cytology  was  established  by  seven  universities,— Harvard, 
Johns  Hopkins,  Columbia,  Pennsylvania,  Michigan,  Cornell  and  Chicago.  It  has  an  editorial 
board  of  10,  viz. 

I^ewellys  F.  Barker,  Chicago  University ;  Thomas  Dwight,  Harvard  University ;  Simon  H. 
Gage,  Cornell  University  ;  G.  Carl  Huber,  Michigan  University  ;  George  S.  Huntington,  Columbia 
University  ;  J.  Playfair  McMurrich,  Univ.  Michigan  ;  Franklin  P.  Mall,  Johns  Hopkins  Univer- 
sity ;  Charles  S.  Minot,  Harvard  University ;  George  A.  Piersol,  Univ.  Pennsylvania  ;  Henry 
McE.  Knower,  Secretary,  Johns  Hopkins  University.  There  are  also  over  60  collaborators  from 
different  institutions. 

The  Journal  of  Comparative  Neurology  and  Psychology,  Granville,  O.  1900+.  This  was 
originally  the  Journal  of  Comparative  Neurology.  It  has  been  reorganized  and  now  has  an 
editor,  Dr.  C.  I,.  Herrick.  a  managing  editor,  C.  Judson  H  errick,  of  Denison  University,  and  two 
associate  editors.  O.  S.  Strong  of  Columbia  University,  and  Robert  M.  Yerkesof  Harvard  Univer- 
sity. Its  collaborators  represent  20  other  institutions. 

The  Journal  of  Experimental  Zoology.  Baltimore,  1904+.  This  new  journal  has  u  editors 
representing  6  Universities  :  Johns  Hopkins,  2, — W.  K.  Brooks  and  R.  G.  Harrison  ;  Harvard 
University,  2, — G.  H.  Parker  and  W.  E.  Castle ;  University  of  Pennsylvania,  2, — E.  G.  Conklin 
and  H.  S.  Jennings  ;  Columbia  University,  i, — E.  B.  Wilson  ;  Chicago  University,  2,  C.  O.  Whit- 
man and  C.  B.  Davenport ;  Bryn  Mawr  College,  i,  T.  H.  Morgan.  Besides  the  editors  there 
are  many  collaborators  from  different  universities. 


INDEX 


Abbe  apertometer,  270-272. 

Abbe  camera  lucida,  123-132. 

Abbe  condenser  or  illuminator,  45-50. 

Abbe's  test-plate,  method  of  using,  268- 
270. 

Aberration,  chromatic,  4,  5  ;  by  corer- 
glass,  55  ;  spherical,  4,  5,  268. 

Absorption  spectra,  136-139,  145-150. 

Acetelyne  light,  37,  51,  229. 

Achromatic  condenser,  42-43,  256  ;  objec- 
tives, u,  64  ;  oculars,  22. 

Achromatism,  12. 

Actinic  focus,  226  ;  image,  221. 

Adjustable  objectives,  n,  12,  54-57;  ex- 
periments. 54  ;  and  micrometry,  118  ; 
and  photo-micrography,  234. 

Adjusting  collar,  graduation  of,  55. 

Adjustment,  of  analyzer,  151  ;  .coarse  or 
rapid,  and  fine,  63,  Frontispiece  ;  of 
objective,  n,  12,  54;  of  objective  for 
cover-glass,  specific  directions,  55  ; 
testing,  63. 

Aerial  image,  30,  31,  230. 

Air  bubbles,  94,  95. 

Albumen  fixative,  Mayer's,  198. 

Alcohol,  absolute,  198 ;  ethylic,  198  ; 
methyl,  198,  203. 

Alcoholic  dye,  staining  sections  with, 
189. 

Alum  solution,  198. 

Amici  prism,  134,  140. 

Amplifier,  109. 

Amplification  of  microscope,  103. 

Analyzer,  141,  151. 

Anastigmatic  objective,  208,  212. 

Angle,  of  aperture,  15,  16 ;  of  carbons  for 
arc  lamps,  251-252  ;  critical,  54. 

Angstrom  and  Stokes'  law  of  absorption 
spectra,  138. 

Angular  aperture,  15,  16. 

Anisotropic,  152. 

Apertometer,  Abbe's,  270. 

Aperture  of  objective,  15,  21,  270;  angu- 
lar, 15,  16  ;  formula  for,  16-18  ;  of 
illuminating  cone,  45  ;  numerical,  17, 
19,  270  ;  numerical  of  condenser,  44  ; 
significance  of,  20. 

Aphengescope,  267. 

Aplanatic  cone,  46  ;  objectives,  n  ;  ocu- 
lar, 22  ;  triplet,  7. 


Apochromatic  condenser,  42  ;  objectives, 
12,  64,  214. 

Apparatus  and  material,  i,  34,  90,  103, 
122,  134,  205,  243  ;  for  micro-projec- 
tion, 256,  257  ;  for  photography,  233  ; 
for  photo-micrography,  205. 

Appearances,  interpretation,  90-102. 

Arc  lamp,  37,  229,  250-255  ;  continuous 
current,  249. 

Arrangement  of  condenser,  46  ;  of  lamp, 
bull's  eye  and  microscope,  51  ;  mi- 
nute objects,  204;  serial  sections, 
192  ;  tissue  for  sections,  191. 

Artifacts,  91. 

Artificial  illumination,  37,  48,  50 ;  for 
photo-micrography,  229. 

Avoidance  of  diffusion  currents,  180 ;  of 
distortion,  124. 

Axial  light,  36 ;  experiments,  40  ;  with 
Abbe  illuminator,  48. 

Axial,  point,  15  ;  ray,  36. 

Axis,  optic,  2,  3,  10  ;  of  illuminator,  49  ; 
secondary,  3,  6,  49. 


!  Back-ground  for  photographing,  209. 
Back  combination  or  system  of  objective, 

9-12. 

I  Bacterial  cultures,  photographing,  241- 
242. 

Balsam,  199 ;  bottle,  183  ;  mounting  in, 
183,  190 ;  preparation  of,  199  ;  re- 
moval from  lenses,  61  ;  nat.ural,  190, 
199 ;  neutral,  199 ;  removal  from 
slides,  162  ;  xylene,  199. 

Bands,  absorption,  137. 

Base  of  microscope,  Frontispiece. 

Bath,  water,  255. 

Bed,  camera,  207. 

Benzin,  removing  from  sections,  189. 

Biaxial  crystals,  154. 

Bibliography,  26,  33,  99,  101,  121,  133, 
150.  I55>  157.  158,159,  196,  204,  220, 
240,  242,  251,  258,  267,  273,  274,  282. 

Blocks  for  collodion,  178  ;  for  shell  vials, 
174. 

Blood,  absorption  spectrum  of,  146  ;  cir- 
culation of  with  micro-projection, 
257,  264. 

Board,  circulation,  280. 

Body  of  microscope,  Frontispiece. 


290 


INDEX 


Bottle  for  balsam,    glycerin   or  shellac, 

172  ;  reagent,  179. 
Bowl,  waste,  181. 
Box,  glass,  181  ;  slide,  198. 
Brownian  movement,  99. 
Bubble,  air,  94,  95. 
Bull's-eye,  51,  217,  227  ;    engraving  glass 

for,  228. 

Burning  point,  7,  30. 
Buxton's  photo-micrographic  outfit,  237- 

238. 


Cabinet  for  microscopical  preparations, 
196,197. 

Calipers,  micrometer,  164  ;  pocket,  164. 

Camera,  bed,  207  ;  for  embryos,  211  ;  for 
large,  transparent  sections,  215  ; 
photo-micrographic,  222,  225,  235  ; 
testing,  223  ;  vertical,  205-210. 

Camera  lucida,  Abbe,  122-132  ;  Wollas- 
ton's,  107,  125. 

Canada  balsam,  199  ;  mounting  in,  183, 
190 ;  preparation  of,  199 ;  removal 
from  lenses,  6r  ;  removal  from  slides, 
162. 

Carbol-xylene,  200. 

Carbon-monoxide  hemaglobin,  spectrum 
of,  147. 

Carbons,  for  projection  apparatus,  250- 
255  ;  adjusting,  251  ;  angle,  251  ; 
kinds,  253  ;  positive  and  negative, 
252  ;  size,  254  ;  wear,  253. 

Card,  catalog,  196  ;  centering,  169  ;  inven- 
tory, 281. 

Care  of,  eyes,  61  ;  micro-projection  appa- 
ratus, 265  ;  microscope,  mechanical 
parts,  59  ;  optical  parts,  60  ;  nega- 
tives, 211  ;  water  immersion  objec- 
tives, 58. 

Carmine,  to  show  currents  and  pedesis, 
99  ;  spectrum  of,  148. 

Carrier,  objective,  259. 

Castor-xylene  clarifier,  200. 

Cataloging,  formula,  195  ;  preparations, 
194-196. 

Cedar- wood  oil,  bottle  for,  199  ;  for  clear- 
ing, 184-185,  200  ;  for  oil  immersion 
objectives,  200. 

Cells,  deep,  thin,  168  ;  isolated,  prepara- 
tion of,  175  ;  mounting,  168  ;  stain- 
ing, 174. 

Cement,  shellac,  203. 

Cementing  collodion,  201. 

Center,  optical,  2,  3. 

Centering,  and  arrangement  of  illumina- 
tor, 43,  47  ;  card,  169  ;  image  of  source 
of  illumination,  44  ;  the  radiant,  255. 

Centimeter  rule,  104,  133. 


Central  .light,  36,  95. 
Chamber,  moist,  171. 

Cheese-cloth  for  cleaning  slides,  161. 

Chemical  focus,  12  ;  rays,  12. 

Chemistry,  Micro- 155. 

Chromatic,  aberration,  4  ;  correction,  n, 
12  ;  correction,  test  for,  270. 

Circulation^  of  blood  with  micro-projec- 
tion, 257,  264  ;  board,  226. 

Clarifier,  200  ;  castor-xylene,  200. 

Class  demonstrations  in  histology  and 
embryology,  243. 

Cleaning  back  lens  of  objective,  61  ;  ho- 
mogeneous objectives,  59 ;  micro-pro- 
,  jection  apparatus,  265  ;  mixtures  for 
glass,  165  ;  sildes  and  cover-glasses, 
161-163  ;  water  immersion  objectives, 
58. 

Clearer,  173,  183,  190,  200. 

Clearing  mixture,  preparation  of,  200 ; 
tissues  with  cedar- wood  oil,  184. 

Clinical  microscope,  243-245. 

Cloudiness  of  objective  and  ocular,  how 
to  determine,  92  ;  removal  60. 

Coarse  adjustment  of  microscope,  Front- 
ispiece ;  testing,  63. 

Cob- web  micrometer,  117. 

Collecting  can,  280 

Collodion,  200  ;  for  coating  glass  rod,  97  ; 
cementing,  201  ;  clarifying,  178  ;  cot- 
ton, 200 ;  for  fastening  sections  to 
slide,  1 88  ;  hardening,  178  ;  method, 
176  ;  thick,  thin,  177,  201. 

Color,  correct  photography,  217  ;  correc- 
tion, 12  ;  images,  54,  58  ;  law  of,  138  ; 
production  of,  154  ;  screens,  218,  220. 

Colored  minerals,  absorption  spectra  of, 
149  ;  specimens,  photography  of,  218. 

Comparison  prism,  141,  142  ;  spectrum, 
142. 

Compensating  ocular,  24,  25. 

Complementary  spectra,  139. 

Compound  microscope,  see  under  micro- 
scope. 

Concave  lenses,  3  ;  mirror,  use  of,  37. 

Condenser,  41-50;  Abbe,  46,  48-49  ;  ach- 
romatic;  42,  43,  227,  256  ;  apochro- 
matic,  42,  227  ;  bull's-eye,  51,  227, 
228  ;  centering,  43,  47  ;  illuminating 
cone  with,  45  ;  lamp,  for  projection, 
250  ;  mirror  for,  48  ;  non-achromatic, 
46 ;  numerical  aperture  of,  44-46 ; 
optic  axis  of,  43,  47  ;  for  photo-micro- 
graphy, 42,  227,  233  ;  standard  size 
for,  26,  47  ;  substage,  42;  See  also 
illuminator. 

Condensing  lens,  36. 

Cone,  aplanatic,  46  ;  illuminating,  45. 

Conjugate  foci,  4. 

Construction  of  images,  geometrical,  6. 


INDEX 


291 


Continuous,  current  arc  lamp,  249  ;  spec- 
trum, 137. 

Contoured,  doubly,  97. 

Converging  lens,  3-5  ;  lens  system,  9. 

Convex  lenses,  3-5. 

Cooler,  specimen,  257. 

Correction,  chromatic,  or  color,  5,  12,  268- 
270  ;  cover-glass,  55-56  ;  cover,  tube- 
length  for,  56-57  ;  over  and  under,  12. 

Cotton,  collodion,  gun,  or  soluble,  200. 

Counterstaining,  189-190. 

Cover- glass,  or  covering  glass,  162-163  \ 
aberration  by,  54  ;  adjustment,  spe- 
cific directions,  55  ;  adjustment  for, 
in  photo-micrography,  234 ;  adjust- 
ment and  tube-length,  12,  13,  55  ; 
anchoring,  170  ;  cleaning,  162-163  ; 
correction,  55,  56  ;  effect  on  rays  from 
object,  17,  55  ;  gauges,  164-165  ;  meas- 
urer, 164-165;  measuring  thickness  of, 
163  ;  non-adjustable  objectives,  table 
of  thickness,  14;  No.  i,  variation  of 
thickness,  164;  putting  on,  94,  167; 
sealing,  169,  170;  size  of,  164,  162; 
thickness  of,  13,  14,  163-165,  193  ; 
tube-length  with,  13,  56,  57  ;  wiping, 
163  ;  with,  serial  sections,  193. 

Crater  of  carbons,  252. 

Critical  angle,  54. 

Crystals,  biaxial,  depolarizing,  154;  from 
frog  for  pedesis,  100. 

Crystallization  under  microscope,  50,  157. 

Crystallography,  155  ;  list  of  substances 
for,  156-157. 

Currents,  diffusion,  avoidance  of,  180 ; 
in  liquids,  98. 

Cutting  sections,  178,  186,  191-192. 


Dark-ground  illumination,  37,  49-50 ; 
with  Abbe  illuminator,  50 ;  with 
mirror,  50. 

Daylight,  lighting  with,  35. 

Deck-plugs  for  collodion  blocks,  178. 

Dehydration,  177,  190. 

Demonstration ,  microscope,  243-245  ; 
with  micro-projection  apparatus,  265. 

Depolarizing  crystals,  154. 

Designation,  of  oculars,  25  ;  of  wave 
length;  143. 

Determination,  of,  field  of  microscope, 
28  ;  equivalent  focus,  272-273  ;  mag- 
nification, 103-109,  273  ;  of  working 
distance,  39. 

Diamond,  writing,  196. 

Diaphragms  and  their  employment,  36-50. 

Diffraction,  grating,  137  ;  illusory  ap- 
pearances due  to,  101. 

Diffusion  currents,  avoidance  of,  180. 


Direct,  light,  35 ;  vision  spectroscope, 
134- 

Dispersing  prism,  137. 

Dissecting  microscope,  8,  33,  175,  228. 

Dissociator,  formaldehyde,  201  ;  nitric 
acid,  203. 

Distance,  principal  focal,  3.  30;  standard 
at  which  the  virtual  image  is  meas- 
ured, 109  ;  working  d.  of  simple  mi- 
croscope or  objective,  39  ;  working 
d.  of  compound  microscope,  n,  34, 
39-40. 

Distinctness  of  outline,  96. 

Distortion  in  drawing,  avoidance  of,  124. 

Diverging  lens,  3. 

Double  spectrum,  142  ;  vision,  103,  105. 

Doubly  contoured,  97  ;  refracting,  152. 

Draw-tube,  Frontispiece  ;  pushing  in,  38. 

Drawing,  with  Abbe  camera  lucida,  129- 
.131  ;  board  for  Abbe  camera  lucida, 
129,  130;  distortion,  avoidance  of, 
124  ;  embryograph  for,  132  ;  with 
microscope,  122;  photographic  cam- 
era for,  132 ;  for  photo-engraving, 
273  ;  scale  and  enlargement  of,  131  ; 
with  simple  microscope,  132. 

Dry  objectives,  10,  16-18  ;  light  utilized, 
17  ;  dry  mounting,  167  ;  numerical 
aperture,  16  ;  dry  plates,  discovery  by 
Maddox,  221. 

Dust,  of  living  rooms,  examination  of, 
101  ;  on  objectives  and  oculars,  how 
to  determine,  92  ;  removal,  60. 

Dye,  general,  staining  with,  189  ;  aque- 
ous, 180,  189  ;  alcoholic,  180,  189. 


Eccentric  diaphragm,  44,  49-50. 

Egg  pippett,  280. 

Electric  light,  37,  229,  235,  250. 

Embryograph,  132 

Embryos,  .camera  for,  211  photograph- 
ing, 211-214;  records  of,  213. 

Engraving  glass  for  bull's-eye  condenser, 
228 

Enlargements,  241. 

Eosin,  201. 

Epidiascope,  266-267. 

Episcope,  267. 

Equivalent  focal  length  or  focus  of  objec- 
tives and  oculars,  10,  25,  272. 

Erect  image  i. 

Ether,  alcohol,  201  ;  sulphuric,  201. 

Ethylic  alcohol,  198. 

Examination  of  dust  of  the  living  rooms, 
bread  crumbs,  corn  starch,  fibres  of 
cotton,  linen,  silk,  human  and  ani- 
mal hairs,  potatoes,  rice,  scales  of 
butterflies  and  moths,  wheat,  101. 


INDEX 


Experiments,  Abbe  condenser,  48  ;  with 
adjustable  and  immersion  objectives, 
54  ;  compound  microscope,  26  ;  ho- 
mogeneous immersion  objective,  58  ; 
lighting  and  focusing,  37  ;  in  micro- 
chemistry,  157  ;  with  micro  spectro- 
scope, 145  ;  with  micro-polariscope, 
152  ;  in  mounting,  166  ;  photo-micro- 
graphy, 229  ;  simple  microscope,  6. 

Exposure,  of  photographic  plates,  232, 
235,  240;  with  color-screen  220. 

Extraordinary  ray  of  polarized  light,  150. 

Eye  and  microscope,  6,  9,  32. 

Eyes,  care  of,  6r  ;  muscae  volitantes  of, 

100. 

Eye-lens  of  the  ocular,  22. 
Eye-piece,  22  ;  micrometer,  1 14. 
Eye-point,   7,  22,  123  ;  of  ocular,  demon- 
stration, 32. 
Eye-shade,  adjusting,  62  ;  double,  62. 


Farrants'  solution  in  mounting  objects, 
171  ;  preparation  of,  201. 

Fibers,  examination  of,  101  ;  textile,  158. 

Field,  28  ;  with  camera  lucida,  107  ;  illu- 
mination of,  45,  51  ;  with  orthoscopic 
ocular,  23 ;  with  periscopic  ocular, 
24  ;  of  view  with  microscope,  28,  29, 
105,  123-125;  size  of,  with  different 
objectives  and  oculars,  28,  29. 

Field-lens,  of  ocular,  22  ;  action  of,  32  ; 
dust  on,  92. 

Filar,  micrometer  ocular,  23,  26  ;  ocular 
micrometer,  117,  118. 

Filter,  hot,  185. 

Filtering  balsam,  etc.,  185,  199. 

Fine   adjustment,  Frontispiece  ;   testing, 

63- 

Fir,  balsam  of,  199. 

Fixative,  albumen,  Mayer's,  198. 

Fixing,  reagents  for,  198  ;  tissue,  176. 

Focal  distance,  or  point,  principal,  30 ; 
length  equivalent,  10. 

Focus,  6;  actinic,  226;  chemical,  12  ; 
conjugate,  4  ;  equivalent,  of  object- 
ives and  oculars,  10,  25,  272-273  ;  op- 
tical, 12  ;  principal^,  5  ;  virtual,  3  ; 
visual,  226. 

Focusing,  6,  34  ;  adjustments,  testing,  63; 
with  compound  microscope,  34  ;  em- 
bryos, 213  ;  experiments,  38  ;  glass, 
209  ;  with  high  objectives,  38  ;  with 
low  objectives,  38  ;  objective  for  mi- 
cro-spectroscope, 144  ;  for  photo-mi- 
crography, 206,  213,  230;  scale  for, 
206  ;  screen  for  photo-micrography, 
209  ;  with  simple  microscope,  6,  34  ; 
slit  of  micro-spectroscope,  145. 


Food,  detection  of  adulteration  in,  158. 

Form  of  objects,  determination  of,  93. 

Formal,  201. 

Formaldehyde,  dissociator,  201  ;  for  isola- 
tion, 173. 

Formula,  for  aperture,  16,  17  ;  for  catalog- 
ing) *95  i  f°r  refraction,  52-54. 

Fraunhofer  lines,  137. 

Free,  hand  sections,  274  ;  working  dist- 
ance, 40. 

Front  combination  or  lens  of  objective, 
9-11. 

Frontal  sections,  192. 

Function  of  objective,  29-31  ;  of  ocular, 


Gauge,  cover-glass,  164-165. 

Gelatin,  liquid,  preparation  of,  203. 

Geometrical  construction  of  images,  6. 

Glass,  cleaning  mixture  for,  165  ;  focus- 
ing, 209  ;  ground,  29,  209  ;  rod  appear- 
ance under  microscope,  96,  97  ;  slides 
or  slips,  161,  162. 

Glasses,  opera,   262. 

Glue,  liquid,  preparation  of,  203. 

Glycerin,  bottle  for,  183  ;  mounting  ob- 
jects in,  order  of  procedure,  170,  201  ; 
removal,  61. 

Glycerin  jelly,  mounting  objects  in,  or- 
der of  procedure,  170-171  ;  prepara- 
tion of,  201. 

Grating,  diffraction,  137. 

Ground  glass,  focusing  screen,  209  ;  pre- 
paration of,  29. 

Gun  cotton,  200. 

H 

Half-tones  from  photo-micrographs,  232, 

274. 
Hardening   collodion,  178 ;     tissue,     176, 

183, 

Hematein,  203. 
Heinatoxylin,  202  ;  stained  preparations, 

photographing,  218. 
Hemoglobin  spectrum,  147. 
High  school  microscope,  64,  71-89. 
Histology,  physiological,  196. 
History  of  photo- micrography,  220. 
Holder,  lens,  7,  217,  228  ;    needle,   167  ; 

slide,  1 88. 
Homogenous   immersion   objective,     16- 

19  ;   cleaning,  59  ;   experiments,  58  ; 

numerical  aperture,  16-21. 
Homogenous  liquid,    n  ;    tester  for,  58, 

271  ;  vessel  for,  199. 
Horizontal  camera,  230,  236-237. 
Huygenian  ocular,  22,  24,  32. 


INDEX 


293 


Illuminating,  cone  (for  condenser),  aper- 
ture of,  45  ;  objective,  13,  238 ; 
power,  21. 

Illumination,  for  Abbe  camera  lucida, 
129;  artificial,  37,  48,  50;  artificial 
for  photo-micrography,  229  ;  center- 
ing image  of  source  of,  44  ;  with  air 
and  oil,  94,  95 ;  dark  ground,  49, 
50  ;  daylight,  35  ;  of  entire  field,  51  ; 
lamp  for,  50  ;  methods  of,  35,  49  ;  for 
micro-polariscope,  151  ;  for  micro- 
spectroscope,  144 ;  oblique  with  air 
and  oil,  94,  95  ;  of  opaque  objects, 
144,  238  ;  for  photography,  208, 
216,  229,  241  ;  for  photo-micrography, 
23°.  233.  238  ;  for  projection.  250  ;  for 
Wollaston's  camera  lucida,  124. 

Illuminator,  41-50;  vertical,  13,  238. 
See  also  condenser. 

Image,  actinic, 221  ;  aerial,  30,  230  ;  center- 
ing i.  of  source  of  illumination,  44  ; 
color,  52,  58  ;  inverted,  real  of  ob- 
jective, 30 ;  of  flame,  45  ;  geomet- 
rical construction  of,  6  ;  and  object, 
size  and  position,  5,  9,  108  ;  real,  5,  9, 
30-32,  103  ;  refraction,  52,  58  ;  retinal, 
6,  9,  32  ;  swaying  of,  48 ;  virtual  i. 
and  standardd'istance  at  which  meas- 
ured, 6,  109. 

Image-power  of  objectives,  18. 

Imbedding,  177,  185. 

Immersion,  fluid  or  liquid,  58,  271  ;  illu- 
minator, 47  ;  objective,  n,  58-59. 

Incandescence  or  line  spectra,  137. 

Incident  light,  35. 

Index,  of  refraction,  53  ;  of  medium  in 
front  of  objective,  16-19. 

Indicator  ocular,  247. 

Infiltration,  collodion,  177  ;  paraffin,  184; 
paraffin  dish  for,  279. 

Ink  for  labels  and  catalogs,  195  ;  for 
drawing,  274. 

Interpretation  of  appearances  under  the 
microscope,  90-102. 

Inventory  card,  281. 

Iris  diaphragm,  82,  157. 

Irrigating  with  reagents,  170. 

Isochromatic  plates,  217. 

Isolation,  173  ;  with  formaldehyde,  173  ; 
nitric- acid,  175. 

Isotropic,  152. 

J-K 

Japanese  filter  or  tissue  paper,  60. 
Jelly,  glycerin,  170-171,  201. 
Jena  glass,  71. 

Jurisprudence^  micrometry  in,  121. 
Knife  support,  277. 


Labels  and  catalogs,  194-196,  203. 

Labeling  microscopical  preparations,  194; 
photographic  negative,  211  ;  serial 
sections.  193. 

Laboratory  compound  microscope,  64, 
71-89. 

Lamp,  acetylene,  37,  51,  229  ;  condenser, 
250  ;  electric  arc,  37,  229,  250-255  ; 
petroleum,  37,  50-51,  220,  229  ;  spirit, 
183. 

Lantern,  magic,  249  ;  slides,  241. 

Law  of  color,  138. 

Lens,  concave,  3  ;  converging,  3  ;  convex, 
4  ;  eye,  22  ;  field,  32  ;  holder,  7,  175, 
217,  228 ;  paper,  60 ;  system,  9  ; 
thick.  3. 

Lenses  of  micro-projection  apparatus, 
cleaning,  265. 

Letters  in  stairs,  93  ;  for  photo-engraving, 
274. 

Lettering  oculars,  26. 

Light,  with  Abbe  illuminator,  48  ;  ace- 
tylene, 37,  51,  229  ;  artificial,  37,  50, 
229 ;  axial,  36,  40,  48  ;  direct,  35  ; 
central,  36,  40  ;  electric,  37,  229,  250  ; 
incident,  35  ;  with  mirror,  37  ;  ob- 
lique, 36,  41,  48  ;  petroleum,  37,  50, 
220,  229  ;  for  photo-micrography,  229; 
polarized,  150 ;  reflected,  35  ;  sun, 
229 ;  transmitted,  36  ;  utilized  with 
different  objectives,  i  7;  for  vertical 
illuminator,  239 ;  wave  length  of, 
142  ;  Welsbach,  37,  229. 

Lighting,  35  ;  for  Abbe  camera  lucida, 
129  ;  artificial,  50  ;  experiments,  37  ; 
with  horizontal  camera,  230  ;  for  mi- 
cro-polariscope, 151  ;  for  micro-spec- 
troscope, 144  ;  with  a  mirror.  37,  40  ; 
with  daylight,  35,  229  ;  for  photog- 
raphy, 208,  216 ;  for  photo-micro- 
graphy, 229,  230 ;  for  vertical  illum- 
inator, 229. 

Line  spectrum,  137. 

Liquid,  currents  in,  98  ;  homogenous,  n, 
58,  271. 

M 

Magic  lantern,  249. 

Magnification  of  compensating  oculars, 
24  ;  effect  of  adjusting  objective,  118  ; 
determination  of,  103-109  ;  expressed 
in  diameters,  103  ;  initial  or  indepen- 
dent, 273 ;  of  microscope,  103 ;  in 
micro-projection,  table,  262  ;  of  mi- 
croscope with  Abbe  camera  lucida, 
131  ;  of  microscope,  compound,  105-; 
of  microscope,  simple,  104  ;  of  photo- 


294 


INDEX 


micrographs,  determination  of,  233  ; 
real  images,  103  ;  table  of,  with  ocu- 
lar micrometer,  in  :  with  projection 
microscope,  262  ;  varying  with  com- 
pound microscope,  109  ;  and  velocity, 
98. 

Magnifier,  tripod,  7,  104,  209. 

Marker  for  preparations,  65-66. 

Marking  objects,  65-66,248  ;  negatives, 
211,  214  ;  objectives,  71. 

Masks  for  preparations,  264. 

Material  and  apparatus,  i,  34,  90,  103, 
123,  I34,_  156,  198,  205,  223,  243. 

Measure,  unit  of,  in  micrometry,  112  ;  of 
wave  length,  143. 

Measurer,  cover-glass,  164-165. 

Measuring  the  thickness  of  cover-glass, 
163. 

Mechanical  parts  of  compound  micro- 
scope, 64  ;  Frontispiece,  8  ;  of  micro- 
scope, care  of,  59  ;  testing,  63. 

Mechanical  stage,  65,  67-70,  258,  259. 

Megascope,  267. 

Metallic  surfaces,  photography  of,  235- 
240  ;  preparation  of,  239. 

Metallography,  microscope  in,  159. 

Metals,  examination  of,  159,  235. 

Met-hemaglobin,  spectrum  of,  136,  147. 

Methods,  collodion,  176-183  ;  paraffin, 
183-191. 

Metric  measures  and  equivalents,  cover 
ist  p.,  133. 

Micro-chemistry,  155-157  ;  slides  for,  161. 

Micro-metallography,   objects  for,  238. 

Micrometer,  103  ;  calipers,  164  ;  cob-web, 
117;  filar  m.  ocular,  117,  nS;  filling 
lines  of,  106 ;  lines,  arrangement  of 
ocular  and  stage,  120  ;  lines,  finding, 
106  ;  net,  128  ;  object  or  objective, 
105;  ocular  or  eye-piece,  114-120; 
ocular,  micrometry  with,  116  ;  ocular, 
ratio,  119;  ocular,  valuation  of,  in, 
114;  ocular,  varying  valuation  of, 
118  ;  for  photo-micrography,  233  ; 
screw  ocular,  117;  stage,  105,  106  ; 
table  of  magnification,  in. 

Micrometry,  definition,  112,  114;  with 
adjustable  objectives,  118;  compari- 
son of  methods,  119-120  ;  with  com- 
pound microscope,  112;  and  juris- 
prudence, 121  ;  limit  of  accuracy  in, 
120;  with  ocular  micrometer,  116  ; 
with  simple  microscope,  112  ;  remarks 
on,  118  ;  unit  of  measure  in,  112. 

Micro-millimeter,  113. 

Micron,  113  ;  for  measuring  wave  length 
of  light,  143. 

Micro-photograph,  220. 

Micro-photography,  distinguished  from 
photo-micrography,  220. 


Micro-planar  objective,  212,  260. 

Micro-polariscope,  100,  150-155. 

Micro-polarizer,  150. 

Micro-projection,  249-267  ;  apparatus, 
256-257  ;  carbons  for,  250-254  ;  cir- 
culation of  blood  with,  257,  264  ;  con- 
denser, 254,  256  ;  current,  254  ;  dem- 
onstration with,  265  ;  magnification, 
262  ;  masks  for  specimens,  264  ;  me- 
chanical stages,  258-259  ;  microscope 
for,  249  ;  objectives  and  oculars,  259  ; 
pointer  for,  265  ;  preparations,  263  ; 
screen  and  screen  distance,  261  ; 
specimen  cooler,  257  ;  stage,  258 ; 
stains  for,  263  ;  water-bath,  255. 

Microscope,  definition,  i  ;  amplification 
of,  103  ;  clinical,  243  ;  demonstration, 
243  ;  dissecting,  8,  33,  175,  176  228  ; 
care  of,  59;  eye  and,  i,  6,  9;  field  of,  28, 
29  ;  focusing,  34  ;  magnification,  103; 
for  metallography,  159-160  ;  for  mi- 
crochemical  analysis,  153;  for  opaque 
objects,  235-237  ;  for  photo-micro- 
graphy, 225,  231,  237  ;  polarizing,  150; 
preparation,  with  erecting  prism  176; 
projection,  249-250  ;  price  of,  64,  71  ; 
putting  an  object  under,  27  ;  sun  or 
solar,  249  ;  screen,  59  ;  stand  for 
large,  transparent  objects,  215,  216  ; 
stand,  for  embryos,  212  ;  traveling, 
245-246. 

Microscope  compound,  definition,  8  ; 
drawing  with,  122  ;  figures,  frontis- 
piece, 9,  71-89,  I02»  J53>  160,  237, 
244-246  ;  focusing,  38-39  ;  for  High 
schools,  64,  71  ;  for  laboratory,  64, 
71-89;  lamp  for,  50;  magnification 
or  magnifying  power,  105  ;  magnifi- 
cation and  size  of  drawing  with 
Abbe  camera  lucida,  131  ;  mechani- 
cal parts,  64  ;  micrometry  with,  112  ; 
optic  axis  of,  9,  10  ;  optical  parts  of, 
9,  64  ;  varying  magnification,  109  ; 
working  distance  of,  39  ;  testing,  63. 

Microscope,  simple,  definition,  i  ;  exper- 
iments with,  6  ;  figures,  6-8,  33,  104, 
175,  209,  228,  243  ;  focusing  with,  34; 
magnification  of,  104  ;  micrometry 
with,  112;  working  distance  of,  34; 

Microscopic,  objective,  9  ;obj  ective  low, 
attached  to  camera,  214  ;  objects, 
drawing,  122  ;  ocular,  22  ;  slides  or 
slips,  161  ;  tube-length,  13,  14. 

Microscopical  preparations,  cabinet  for, 
197  ;  cataloging,  194  ;  labeling,  194  ; 
mounting,  166-193. 

Microtome,  hand,  274-275  ;  Minot's  275- 
.276  ;  razor  support  for,  276-277  ;  slid- 
ing, 278. 


INDEX 


295 


Micro-spectroscope,    134-150;   adjusting, 
139 ;     experiments,     145 ;     focusing, 
144  ;  focusing  the  slit,  139 ;  lighting 
for,  144  ;  objectives  to  use  with,  144  ; 
reversal,  apparent,  of  colors  in,  134  ; 
slit,  mechanism  of,  135,  139. 
Micrum,  113. 
Mikron,  113. 

Milk  globules,  to  overcome  pedesis 
of,  loo 

Minerals,  absorption  spectra  of,  149. 

Minot's  microtomes,  275-276. 

Minute  objects,  arrangement  of  204. 

Mirror,  9-11  ;  for  Abbe  illuminator,  48; 
of  camera  lucida,  arrangement  for 
drawing,  126  ;  concave,  use  of,  37  ; 
dark  ground  illumination,  50  ;  light 
with,  central  and  oblique,  40,  41  ; 
lighting  with,  37  ;  plane,  use  of,  37. 

Mixture,  clearing,  200. 

Models,  wax,  274. 

Moist,  chamber,  171. 

Molecular  movement,  99. 

Monazite  sand,  spectrum  of,  149. 

Mounting  cells,  preparation  of,  168  ;  me- 
dia and  preparation  of,  198  203  ;  ob-  j 
jects    for    polariscope,    151  ;   perma-  j 
nent,  167  ;  temporary,  166. 

Mounting   objects,  dry   in    air,  order  of 
procedure,  167-168;  in   glycerin,  or-  i 
der   of  procedure,    170  ;  in   glycerin  \ 
jelly,    order  of    procedure,    170;    in 
media  miscible  with  water,  169  ;  mi- 
nute   objects,   204 ;  opaque    objects, 
239  ;     permanent,    167  ;   in    resinous  ! 
media,  by  drying  or  desiccation,  or-  j 
der  of  procedure,    172  ;   in   resinous 
media,  by  successive   displacements, 
order  of  procedure,  172  ;  temporary," 
1 66. 

Movement,  Brownian,  or  molecular,  99. 

Muscae  volitantes,  100. 

Muscular  fibers,  isolation  of,  175. 

N 

Natural  balsam,  199. 

Needle-holder,  167. 

Negatives,  labeling,  211,  214;  oculars, 
22  ;  rack  for  drying,  240  ;  record  of, 
214  ;  storing,  211,  214. 

Net  micrometer,  128.    • 

Neutral  balsam,  199. 

Nicol  prism,  150. 

Nitric  acid,  dissociator,  203. 

Nomenclature  of  objectives,  10. 

Non-achromatic  condenser,  46 ;  object- 
ives, ri. 

Non-adjustable  objectives,  12;  thickness 
of  cover  glass  for,  table,  14. 


Normal  salt  solution,  203. 

Nose-piece,  27,  38,  80;  marking  object- 
ives on,  71. 

Numerical  aperture,  of  condenser,  44  ;  of 
objectives,  16,  270  ;  table  of,  19. 


Object,  determination  of  form,  93  ;  hav- 
ing plane  or  irregular  outlines,  rela- 
tive position  in  a  microscopical  prep- 
ration,  92  ;  and  image,  size  of,  10, 
108  ;  marking  parts  of,  65-66  ;  mark- 
ing position  of,  248  ;  micrometer,  105; 
mounting,  166  ;  putting  under  micro- 
scope, 27  ;  shading,  59  ;  suitable  for 
photo-micrography,  227  ;  transparent 
with  curved  outlines,  relative  posi- 
tion in  microscopic  preparations,  94. 

Objective,  9-13,  121  ;  achromatic,  n  ; 
adjustable,  n,  12,  54;  adjustable, 
micrometry  with,  118;  adjustable, 
photo-micrography  with,  234  ;  adjust- 
ment for,  54 ;  aerial  image  of,  30  ; 
anastigmat,  208  ;  aperture  of,  15-22, 
270  ;  aplanatic,  1 1  ;  apochromatic, 
12,  224  ;  back  combination  of,  10,  n  ; 
carrier,  259  ;  cleaning  back  lens  of, 
61  ;  collar,  graduated  for  adjustment, 
56  ;  cloudiness  or  dust,  how  to  deter- 
mine, 92;  designation  of.  10;  dry, 
10,  17-19,  121  ;  equivalent  focus  of, 
10,  25,  29,  272  ;  field  of,  28-29  J  focus- 
ing for  micro-spectroscope,  144  ;  front 
combination  of,  10,  u  ;  function  of, 
29"3T'»  glass  for,  11-13,  71  '•>  high, 
focusing  with,  38  ;  homogeneous  im- 
mersion, 17-19,  121  ;  homogeneous 
immersion,  cleaning,  59;  homogene- 
ous immersion,  experiments,  58  ;  il- 
luminating, 13,  238 ;  image,  power 
of,  18  ;  immersion,  ir,  121  ;  index  of 
refraction  of  medium  in  front  of,  17, 
19  ;  initial  or  independent  magnifica- 
tion of,  273  ;  inverted,  real  image  of, 
30  ;  for  laboratory  microscope,  64  ; 
lettering,  10  ;  light  utilized  with,  17  ; 
low,  focusing  with.  38 ;  magnifica- 
tion of,  272  ;  marking,  by  Krauss' 
method,  71  ;  for  micrometallography, 
13,  159;  micro-planar,  212,  260;  to 
use  with  micro-polariscope,  151  ;  mi- 
croscopic, 9  ;  to  use  with  micro- 
spectroscope,  144  ;  for  micro- spectro- 
scope, focusing,  144  ;  nomenclature 
of,  10 ;  non-achromatic,  n  ;  non- 
adjustable,  12;  non-adjustable,  thick- 
ness of  cover-glass  for,  table,  14 ; 
with  nose  piece,  38  ;  numbering,  10  ; 
numerical  aperture,  16-22,  270 ;  oil 


296 


INDEX 


immersion,  n  ;  panto-chromatic,  13  ; 
para-chromatic,  13  ;  for  photography, 
208,  212,  214,  224;  for  photo-microg- 
raphy, 224;  projection,  13,  212,  214, 
259 ;  putting  in  position  and  remov- 
ing, 26  ;  semi-apochromatic,  13;  table 
of  field,  29  ;  terminology  of,  10  ;  un- 
adjustable,  12;  variable,  13;  visual 
and  actinic  foci  of,  in  photo-microg- 
raphy, 226  :  water  immersion,  17-19, 
56  ;  working  distance  of,  39-40. 

Oblique  light,  36,  41  ;  with  Abbe  illumi- 
nator, 48  ;  with  a  mirror,  41,  50. 

Ocular,  various  forms,  22-25  \  cloudiness, 
how  to  determine  and  remove,  60,  92; 
equivalent  focus  of,  25,  29,  273  ;  eye- 
point  of,  32  ;  field-lens,  32  ;  filar  or 
screw  micrometer,  26,  117;  focus, 
equivalent  of,  25,  273  ;  function  of, 
31-33  ;  indicator,  67,  247  ;  iris  dia- 
phragm for,  157  ;  lettering  and  num- 
bering, 26  ;  micrometer,  micrometry 
with,  114-121  ;  parfocal,  24,  38;  for 
photo-micrography,  224,  232,  235  ; 
pointer,  247  ;  projection,  260  ;  spec- 
troscopic,i34  ;  standard  size  for,  26; 
table,  effect  on  field,  29. 

Oil,  and  air,  appearances  and  distinguish- 
ing optically,  95  ;  removal,  61  ;  re- 
moval from  sections,  179. 

Oil-globules,  with  central  and  oblique 
illuminations,  95. 

Oil  immersion  objectives,  ri. 

Opaque  objects,  lighting,  144,  238  ;  pho- 
tography of,  with  microscope,  235- 
240  ;  projection  of,  267. 

Opera  glasses,  262. 

Optic  axis,  2,  ;  of  condenser  or  illumi- 
nator, 47  ;  of  microscope,  10. 

Optical,  bench,  237  ;  center,  2  ;  focus,  12  ; 
parts  of  compound  microscope,  9,  64; 
parts  of  microscope,  care  of,  and  test- 
ing, 60,  63  ;  section,  98. 

Order  of  procedure  in  mounting  ob- 
jects dry  or  in  air,  167  ;  in  glycerin, 
170;  in  glycerin  jelly,  170:  in  resin- 
nous  media  by  desiccation,  172  ;  in 
resinous  media  by  successive  dis- 
placement, 172. 

Ordinary  ray,  with  polarizer,  150. 

Orthochromatic  plates,  217. 

Orthoscopic  ocular,  field  with,  28. 

Outline  distinctness  of,  96. 

Oven  paraffin,  279. 

Over-correction,  5. 

Oxy-hemoglobin,  spectrum  of,  138,  147. 


Paper,  bibulous,  filter,  lens,  or  Japanese 
for  cleaning  oculars  and  objectives, 
60,  i  So. 

Paraffin,  185,  203  ;  filtering,  185  ;  infil- 
trating with,  185  ;  dish  for  infiltrat- 
ing, 279;  imbedding  in,  185,  186  ; 
method,  183  ;  oven,  279  ;  pail  for 
melting,  184  ;  removal  from  lenses, 
61  ;  removing  from  sections,  188. 

Parfocal  oculars,  24,  38. 

Parts,  optical  and  mechanical  of  micro- 
scope, 8,  64  ;  testing,  63. 

Pedesis,  99  ;  compared  with  currents,  99  ; 
to  overcome,  100 ;  with  polarizing 
microscope,  100 ;  proof  of  reality  of, 

100. 

Penetrating  power,  21. 

Penetration  of  objective,  21. 

Permanent  mounting,  167  ;  preparations 
of  isolated  cells,  175. 

Permanganate  of  potash,  absorption  spec- 
trum of,  136,  146. 

Petri  dish,  photographing  bacterial  cul- 
tures in,  241. 

Petroleum  light,  37,  229  ;  as  color  screen, 
220. 

Pharmacological  products,  examination 
of,  158. 

Photo-engraving,  drawing  for,  273  ;  let- 
tering for,  274. 

Photographic,  camera,  207  ;  negatives, 
labeling,  211,  214  ;  objectives,  208  ; 
prints,  211. 

Photography,  back -ground  for,  209  ;  of 
bacterial  cultures,  241-242  ;  color- 
correct,  217  ;  of  colored  objects,  218  ; 
compared  with  photo-micrography, 
222;  of  embryos,  211-214;  focusing 
and  exposure,  206,  213  ;  indebtedness 
to  photo-micrography,  220;  of  large 
transparent  objects,  214-216;  lighting 
for,  208,  216;  metallic  objects,  235- 
240  ;  objectives  for,  208,  212,  214  ;  of 
objects  in  alcohol  or  water,  206  ; 
opaque  objects,  235-240  ;  plates  for, 
217;  stage  for,  211  ;  with  vertical 
camera,  205-209,  225. 

Photo-micrograph,  220;  determination 
of  magnification  for,  233  ;  at  5-20  di- 
ameters, 212  ;  20-50  diameters,  230  ; 
100-2500  diameters,  233  ;  of  metallic 
surfaces,  235-240 ;  objects  suitable 
for,  227  ;  of  opaque  objects,  235-240  ; 
prints  of,  211  ;  plates  for,  217  ;  repro- 
ductions of,  232  ;  with  and  without 
1  an  ocular,  230-234. 

Photo-micrographic,  camera,  222,  225, 
236  ;  outfit,  236-237  ;  stand,  231. 


INDEX 


297 


Photo-micrography,  220-240 ;  cover-glass 
correction,  234 ;  apparatus  for,  223  ;  i 
compared  with  ordinary  photogra- 
phy, 222  ;  condenser  for,  42,  226,  227;  | 
distinguished  from  micro-photogra- 
phy, 220  ;  experiments,  229  ;  expos- 
ure for,  213,  220,  232,  235,  240  ;  focus- 
ing for,  213,  216  ;  focusing  screen  for, 
209 ;  lighting,  229,  230,  233,  239  ;  mi- 
crometer for,  233  ;  objectives  and  oc- 
ulars for,  13,  224,  232,235  ;  vertical 
camera  with,  211,  222,  225  ;  actinic 
foci  in,  226  ;  with  and  without  ocular, 
230,232,  234  ;  record  table  for,  219. 

Physiological  histology,  196. 

Picric-alcohol,  203. 

Picro-fuchsin,  190. 

Pillar  of  microscope,  Frontispiece. 

Pin-hole  diaphragm,  47. 

Pippett,  179;  egg,  280. 

Plane  mirror,  use  of,  37. 

Plates,  color-correct,  217  ;  exposure  of, 
213,  220,  232,  235,  240  ;  isochromatic, 
or  orthochromatic,  217  ;  size  of,  224. 

Pleochroism,   152. 

Pleurosigma  angulatum,  41. 

Point,  axial,  15  ;  burning,  7. 

Pointer  ocular,  247. 

Pol  ari  scope,  140,  150. 

Polarized  light,  extraordinary  and  ordi- 
nary ray  of,  150. 

Polarizer  and  analyzer,  140,  151. 

Polarizing  microscope,  pedesis  with,  100. 

Position  of  objects  or  parts  of  same  ob- 
ject, 92  ;  marking  p.,  248. 

Positive  oculars,  10,  22. 

Power,  of  microscope,  103  ;  illuminating, 
penetrating,  resolving,  of  objective, 
19-21  ;  of  ocular,  25. 

Preparation  of  Canada  balsam,  Farrant's 
solution,  glycerin,  glycerin  jelly, etc., 
1 98-204. 

Preparation,  of  clearing  mixture,  liquid 
gelatin  and  shellac  cement,  198-204  ; 
of  ground,  glass,  29  ;  of  metallic  sur- 
faces, 239  ;  vials,  174. 

Preparations,  cataloging,  194-196 ;  cabi- 
net for,  196-197  ;  labeling,  194 ;  for 
microprojection,  263  ;  permanent,  of 
isolated  cells,  175. 

Price  of  American  and  foreign  micro- 
scope, 71. 

Principal,  focus,  3,  5  ;  focal  distances,  3, 
30  ;  optic  axis,  2,  5. 

Prism  of  Abbe  camera  lucida,  124,  127; 
Amici,  140 ;  comparison,  141  ;  dis- 
persing, 141  ;  erecting,  176 ;  Nicol, 
150  ;  and  slit  of  micro-spectroscope, 
mutual  arrangement,  139  ;  of  Wollas- 
ton's  camera  lucida,  125. 


Prints,  photographic,  211. 

Projection,  apparatus,  249,  263-297  ;  mi- 
croscope, 249-266  ;  see  micro-projec- 
tion ;  objective,  13,  214,  259  ;  ocular. 
24,  25,  232,  260  ;  opaque  objects,  267  ; 
in  photo-micrography,  232,  234. 

Putting,  on  cover-glass,  "167  ;  an  object 
under  microscope,  27  ;  an  objective 
and  ocular  in  position,  26,  27. 

Pyroxylin,  200. 

Q-R 

Quadrant  for  camera  lucida,  127,  128. 

Radiant,  centering,  255. 

Ratio,  ocular  micrometer,  119. 

Razor  and  support,  276-277. 

Reagent,  bottle,  179  ;  for  fixing,  198;  irri- 
gation with,  170  ;  for  mounting,  198. 

Real  image,  5,  8,  9,  30;  magnification,  103, 
262. 

Record,  of  negatives,  214;  table  for  photo- 
micrography, 219. 

Reflected  light.  35. 

Reflection,  total,  54. 

Refraction,  52  ;  images,  52,  58  ;  index  of, 
53  ;  of  medium  in  front  of  objective, 

19- 

Refractive,  doubly,  152  ;  highly,  97  ; 
singly,  152. 

Relative  position  of  objects.  92. 

Resinous  media,  mounting  objects  in, 
order  of  procedure,  by  drying  or 
desiccation,  172  ;  by  a  series  of  dis- 
placements, 172. 

Resolution  and  numerical  aperature,  20. 

Resolving  power,  20. 

Retinal  image,  6,  9. 

Revolving  nose-piece,  marking  objectives 
on,  71. 

Ribbon   sections,  186-187  ;  trav  f°r»  l87- 


Sagittal  sections,  192. 

Salicylic  acid,  crystallization,  50. 

Salt  solution,  normal,  203. 

Scale,  of  drawing,  131  ;  of  sizes  for  pho- 
tographing, 206 ;  of  wave  lengths, 
142. 

Screen,  color,  218-219;  focusing  s.  for 
photo-micrography,  209 ;  of  ground 
glass,  29;  for  micro-projection,  261  ; 
for  microscope,  59. 

Screw,  society,  64  ;   micrometer,  26,  117. 

Sealing  cover-glass,  169,  170. 

Searching  ocular,  24. 

Secondary  axis,  3. 

Section,  lifter,  181-182  ;  optical,  98. 

Sections,  arrangement  of  tissue  for,  191  ; 
clearing,  190;  cutting,  178,  186 ;  de- 


298 


INDEX 


hydration  of,  190  ;  extending  with 
water,  186  ;  fastening  to  slide,  179, 
187  ;  frontal,  192  ;  mounting,  183, 
190  ;  removing  benzine,  oil  and  para- 
ffin from,  179,  188  ;  ribbon,  186  ; 
sagittal,  192  ;  serial,  191-193  ;  stain- 
ing, 1 80,  189;  transferring,  179; 
thickness  for  micro-projection,  263. 

Selenite  plate  for  polariscope,  154. 

Semi-apochromatic  objective,  13. 

Serial  sections,  191-193  ;  arranging  and 
labeling,  192,  193  ;  stage  for,  67,  69  ; 
thickness  of  cover-glass  for,  193. 

Shell  vials,  174. 

Shellac  cement,  preparation  of,  203  ;  re- 
moval from  lenses,  61. 

Significance  of  aperture,  20. 

Simple  microscope,  see  under  microscope. 

Sines,  table  of,  3d  page  of  cover. 

Slides,  161  ;  box  for  198  ;  cleaning,  161  ; 
holder  for,  188-189  ;  lantern,  241  ;  for 
micro-chemistry,  161  ;  tray  for,  187. 

Sliding  microtome,  278. 

Slips,  161. 

Slit    mechanism    of  micro-spectroscope, 

135,  139- 

Society  screw,  64. 

Sodium,  lines  and  spectrum,  136-137. 

Solar  spectrum  or  s.  of  sunlight,  136-137. 

Soluble  cotton,  200. 

Solution,  alum,  198;  Farrants',  201. 

Specimen  cooler,  257. 

Spectral,  colors,  138  ;  ocular,  134,  139. 

Spectroscope,  direct  vision,  134,  145. 

Spectroscopic,  examination  of  color- 
screens,  220  ;  ocular,  134. 

Spectrum,  136-150;  absorption,  137; 
amount  of  material  necessary  and  its 
proper  manipulation,  145  ;  analysis, 
150  ;  Angstrom  and  Stokes'  law  of, 
138  ;  banded,  not  given  by  all  colored 
objects,  148  ;  of  blood,  146  ;  of  carbon 
monoxide  hemaglobin,  147  ;  of  car- 
mine solution,  148  ;  of  minerals,  149  ; 
of  colorless  bodies,  148  ;  comparison, 
142  ;  complementary,  139  ;  continu- 
ous, 137  ;  double,  142  ;  incandescence; 
137  ;  line,  137  ;  met-hemaglobin,  147, 
monazitesand,  149  ;  oxy-hemoglobin, 
138, 147;  permanganate  of  potash,  136, 
146  ;  single-banded  of  hemaglobin, 
138,  147  ;  sodium,  136,  137  ;  solar, 
J36'  T37  ;  two-banded  of  oxy-hetna- 
globin,  147. 

Spherical   aberration,  4,  5  ;  test  for,  268. 

Stage,  Frontispiece,  65  ;  mechanical,  65, 
67-70,  259  ;  micrometer,  105  ;  for  mi- 
cro-projection, 258  ;  for  photograph- 
ing, 211. 


Stain,  alcoholic,  180,  189;  aqueous,  180, 
189  ;  for  micro-projection,  263. 

Staining,  cells,  174  ;  dish,  190  ;  sections, 
1 80,  189. 

Stand,  of  microscope,  65  ;  photo-micro- 
graphic,  231  ;  special  for  embryos, 
212  ;  special  for  large  transparent  ob- 
jects, 215,  216. 

Standard,  distance  (250  mm.)  at  which 
the  virtual  image  is  measured,  109  ; 
screw,  64  ;  size  for  condenser,  47  ; 
size  for  oculars,  26. 

Stokes  and  Angstrom's  law  of  absorption 
spectra,  138. 

Storing  negatives,  211  ;  preparations,  196. 

Substage,  86,  Frontispiece. 

Substances  for  crystallography,  156. 

Sulphonal  with  polarizer,  154. 

Sulphuric  ether,  201. 

Support  for  knife  of  microtome,  276-277. 

Swaying  of  image  48. 

Synthol,  198. 

System,  back,  front,  intermediate  of 
lenses,  10,  u  ;  crystal,  156;  metric, 
cover  ist  p.,  133. 


Table,  for  immersion  fluid,  272  ;  of  mag- 
nification and  valuation  of  ocular  mi- 
crometer, in  ;  magnification  with 
projection  microscope,  262  ;  of  tube- 
length  and  thickness  of  cover-glasses, 
14  ;  natural  sines,  third  page  of  cover; 
of  numerical  aperture,  19 ;  record, 
for  photo-micrography,  219  ;  size  of 
fields,  29  ;  testing  homogeneous  liq- 
uids, 272  ;  of  valuations  of  ocular 
micrometer,  in  ;  weights  and  meas- 
ures, 2d  page  of  cover. 

Temporary  mounting,  166. 

Terminology  of  objectives,  10. 

Test  of  chromatic  and  spherical  aberra- 
tion, 268-270. 

Tester,  cover-glass,  164-165  ;  for  homo- 
geneous liquids,  58,  271. 

Testing  a  camera,  223  ;  a  microscope  and 
its  parts,  63. 

Test-plate,  Abbe's,  method  of  using,  268. 

Textile  fibers,  examination  of,  101,  158. 

Thickness,  of  cover-glass  for  non-adjust- 
able objectives,  table,  14  ;  of  serial 
sections,  193. 

Tissues,  arranging  for  sections,  191  ;  fix- 
ing or  hardening,  176,  183  ;  washing 
apparatus  for,  280. 

Tolles-Mayall  mechanical  stage,  67. 

Transections,  192.   . 

Transferring  sections,  179. 

Transmitted  light,  36. 


INDEX 


299 


Tray  for  slides,  187. 

Triplet,  aplanatic,  7. 

Tripod,  7,  104  ;  base  for  microscope,  86  ; 
as  focusing  glass,  209. 

Tube  of  microscope,  Frontispiece. 

Tube-length,  13,  14,56,  for  cover-glass 
adjustment,  56,  57  ;  importance  of, 
56  ;  microscopical,  13,  14 ;  of  various 
opticians,  table,  14  ;  and  optical  com- 
binations, 118. 

Turn-table,  168. 

U— V— W— X 

Unadjustable  objectives,  12. 

Under-correction,  5. 

Unit  of  measures,  in  micrometry,  112  ;  of 
wave  length,  143. 

Valuation  of  ocular  micrometer,  114-116  ; 
table,  in. 

Variable  objective,  13. 

Varying  magnification  of  compound  mi- 
croscope, 109. 

Varying  ocular  micrometer  valuation, 
118. 

Velocity  under  microscope,  98. 

Vertical,  camera,  205-210,  225  :  illumina- 
tor, 13,  238-239. 


Vials,  preparation,   174  ;   blocks  for,  174. 

Virtual  image,  5,  6,  9,  32  ;  standard  dis- 
tance at  which  measured,  109. 

Visibility  with  objectives,  20. 

Vision,  double,  103,  105  ;  microscopic,  21. 

Washing  apparatus  for  tissues,  280. 

Waste  bowl,  181. 

Water  immersion  objective,  16-19,  56 ; 
light  utilized,  17  ;  numerical  aper- 
ture, 19. 

Water,  bath,  235,  255  ;  for  immersion  ob- 
jectives and  removal  of,  56,  58. 

Wave  length,  designation  of,  143  ;  scale 
of,  142. 

Wax  models,  274. 

Weights  and  measures,  see  2d  page  of 
cover. 

Welsbach  light,  37,  229. 

Wollaston's  camera  lucida,  107,  125. 

WTork-room  for  photo-micrography,  224. 

WTork-table,  position,  etc.,  62. 

Working  distance  of  microscope  or  ob- 
jective, ii,  34,  39-40. 

WTriting  diamond,  196. 

Xylene,  178,  200  ;  balsam,  199  ;  for  re- 
moving oil,  179 ;  removing  from 
slides,  189. 

Xylol,  German  form  of  xylene,  178,  200. 


GENERAL  LIBRARY 
UNIVERSITY  OF  CALIFORNIA— BERKELEY 

RETURN  TO  DESK  FROM  WHICH  BORROWED 

This  book  is  due  on  the  last  date  stamped  below,  or  on  the 

date  to  which  renewed. 
Renewed  books  are  subject  to  immediate  recall. 


9 1954 


LD  21-100m-l,'54(1887sl6)476 


TABLE   OF   NATURAL  SINES 
Compiled  from  Prof.  G.  W.  Jones'  Logarithmic  Tables 


MINUTES. 

DEGREES  AND 

QUARTER  DEGREES  UP  TO 

90°. 

i'  0.00029    i°, 

1 
0.01745  16°, 

0.2756431°, 

0.5150446°, 

0.7193461°, 

0.87462  76°, 

0.97 

2  000058    i°,i5/ 

0.02181  i6°,i5x 

0.27983  31°,  1  5 

'  0.51877  46°,  15'  0.72236  61°,  15'  0.87673  76°,i5 

'0.97 

3  0.00087    1,30 

0.02618  16,30 

0.28402  31,30 

0.5225046,30 

0.72537  61,30 

0.87882  76,30 

o-97 

4  0.00116    1,45 

0.03054  16,45 

0.2882031,45 

0.52621  46,45 

0.7283761,45 

o  88089  76,45 

0.97 

5  0.00145!  2 

0.03490!  1  7 

0.2923732 

o  52992  47 

0.73135  62 

0.88295  77 

0.97 

6  0.00175:  2,15 

0.03926117,15 

0.2965432,15 

0.53361:47,15 

07343262,15 

0.8849977,15 

o.97 

7  0.00204    2,30 

0.04362  17,30 

0.30071  32,30 

o.5373o|47,30 

0.7372862,30 

0.88701  77,30 

0.97 

8  0.00233    2,45 

0.0479  N  1  7,45 

o  30486  32,45 

0.54097  47,45 

0.74022  62,45 

o  8890277,45 

0.97 

9  o  00262    3 

0.05234  18 

0.30902  33 

o  54464  48 

0.7431463 

0.89101  78 

0.97 

10  0.00291 

3,i5 

00566918,15 

0.3131633,15 

0.5482948,15 

0.7460663,15 

0.89298178,15 

o.97 

ii  0.00320   3,30 

0.06105118,30 

0.3173033,30 

0.5519448,30 

0.74896:63,30 

o  8949378,30 

0-97 

12  0.00349    3,45 

0.06540  18.45 

0.3214433,45 

o.555574«,45 

0.75184163,45 

0.89687  78,45 

0.98 

13  0.00378   4 

0.06976  19 

o  32557  34 

o.559J9  49 

0.75471164 

0.89879  79 

0.98 

14  000407   4,15 

o.o74ii|i9,i5 

0.3296934,15 

0.5628049,15 

0.75756:64,15 

0.9007079,15 

0.98 

15  000436   4,30 

0.07846:19,30 

0.3338134,30 

o  56641  49.30 

0.76041  64,30 

0.9025979,30 

0.98 

1  6  0.00465    4,45 

0.08281  19,45 

0.33792  34,45 

0.5700049,45 

0.7632364,45 

0.9044679,45 

0.98 

17  0.00495    5 

0.0871620 

0.3420235 

0.5735850 

0.76604165 

0.90631  80 

o.gS 

18  0.00524    5,15 

0.09150  20  15 

o.346i2'35,i5 

05771550,15 

0.7688465,15 

0.9081480,15 

0.98 

19  0.00553    5.30 

0.09585120,30 

03502135,30 

0.5807050,30 

0.77162  65,30 

0.90996:80,30 

0.98 

20  0.00582:  5,45 

0.10019  20,45 

o  35429  35,45 

0.58425)50,45 

0.7743965,45 

0.9117680,45 

0.98 

21  0.00611    6 

0.1045321 

0.35837  36 

0.5877951 

0.77715  66 

o.9l335pi 

0.98 

22  0.00640!  6,15 

0.10887  21,15 

036244136,15 

0.5913151,15 

07798866,15 

0.91531  81,15 

0.98 

23  0.00669    6,30 

0.1132021,30 

o  36650  36,30 

0.59482  51,30 

0.78261:66  30 

0.91706  81,30 

0.98 

24  o  00698 

6,45 

0.1175421,45 

0.37056  36,45 

0.5983251,45 

0.7853266,45 

0.9187981,45 

0.98 

25  0.00727 

7 

O.I2I87  22 

0.37461  37 

0.60182  52 

o  78801  67 

o  92050  82 

0.99 

26  0.00756 
27  000785 

7,i5 
7,30 

O.I262O  22,15 

0.1305322,30 

0.37865137,15 
038268:37,30 

0.6052952.15 
0.6087652.30 

0.7906967,15 
o  79335  67,30 

0.9222082,15 
0.92388182,30 

099 
0.99 

28  0.00814 

7,45 

0.1348522,45 

0.38671  37,45 

0.61222  52,45 

o  79600  67,45 

o.92554;82,45 

0.99 

29  0.00844 

8 

0  I39i7'23 

o  39073  38 

0.6156653 

0.79864  68 

0.9271883 

0.99 

30  0.00873 

8,15 

0.1434923,15 

03947438,15 

0.61909153,15 

0.80125  68,15 

0.9288183,15 

0.99 

31  0.00902 

8.30 

0.14781  23,30 

0.39875  38,30 

062251(53,30 

0.8038668,30 

0.93042  83,30 

0.99 

32  0.00931 

8,45 

o  15212  23,45 

o  40275  38,45 

0.62592(53,45 

0.80644!  68,  45 

0.93201  83.45 

0.99 

33  0.00960 

9 

o.  15643  24 

o  40674(39 

0.62932154 

0.8090269 

0.9335884 

0.99 

34  0.00989 

9,i5 

0.16074  24  15 

0.41072:39,15 

0.63271154,15 

0.81157  69  15 

0.93514184,15 

0-99 

35  0.01018 

9.30 

0.16505  24,30 

0.4146939,30 

0.6^608154,30 

0.8141269.30 

0.93667  84,30 

0-99 

36  o  01047 

9,45 

0.16935:24,45 

0.41866  39.45 

0.6394454,45 

0.81664169,45 

0.93819  84,45 

0.99 

37  0.01076 

10 

o  17365125 

0.42262  40 

0.6427955 

0.8191570 

09396985 

0.99 

38  0.01105 

10,15 

0.1779425,15 

04265740,15 

0.6461255,15 

0.82165170,15 

0.9411885,15 

0.99 

39  001134 
40  0.01164 

10,30 
io,45 

0.1822425,30 
0.1865225,45 

0.43051  40,30 
0.43445  4°,45 

0.6494555,30 
0.6527655,45 

0.82413  70,30 
0.82659  70,45 

0.94264185,30 
0.94409  85,45 

0.99 
0.99 

41  0.01193 

ii 

0.19081  26 

0.4383741 

0.6560656 

0.82904  71 

0.94552  86 

0-99 

42    0.01222 

11,15 

0.19509126,15 

0.4422941,15 

0.6593556,15 

0.8314771,15 

0.94693:86,15 

0-99 

43  0.01251 

11,30 

0.19937126,30 

0.4462041,30 

0.66262  56,30 

0.8338971,30 

0.94832  86,30 

0-99 

44  0.01280 

n,45 

0.2036426,45 

0.45010  41,45 

0.66588:56,45 

0.83629  7i,45 

0.949708645 

0.99 

45  0.0130912 
46  0.01338  12,15 

o  20791(27 
0.21218  27,15 

0-4539942 
0.4578742,15 

0.66913157 
0.6723757,15 

0.83867  72 
0.8410472,15 

0.9510687 
0.9524087,15 

0-99 
0.99 

47  0.01367  12,30 

0.21644  27,30 

0.4617542,30 

o.67559i57-3° 

0.84339  72,30 

0.95372  87,30 

0.99 

48  0.01395  12,45 

0.22070^7,45 

0.46561  42,45 

0.67880157,45 

o  84573  72,45 

0.95502  87,45 

0-99 

49  0.01425  13 

0.22495:28 

0.46947  43 

o.682ooi58 

0,84805  73 

0.95630  88 

0.99 

50  0.01454  13,15 

0.2292028.15 

o.47332!43,i5 

0.68^1858,15 

0.8503573,15 

o.95757|88,i5 

0.99 

51  0.01483 

13-3° 

0.2334528,30 

0.47716143,30 

0.6883558,30 

0.8526473.3° 

0.95882188.30 

0.99 

52  0.01513 

1345 

0.2376928,45 

0.48099143,45 

0.69151  58.45 

0.85491  73,45 

o  96005  88,45 

0.99 

53  0.01542 

14 

0.24192129 

0.48481144 

0.69466  59 

0.85717  74 

0.9612689 

0.99 

54  0.01^7! 

14.15 

0.24615  29,15 

048862144,15 

0.6977959,15 

0.85941  74,15 

0.9624689,15 

o.99< 

55  o.oi6o0 

14,3° 

0.25038:29,30 

0.49242  44,30 

0.70091:59,30 

0.86163  74,3° 

0.9636389,30 

0-99 

56  0.01629  14,45 

0.254602945 

0.49622  44,45 

o  70401  59,45 

0.8638474,45 

0.9647989,45 

0-99 

57  0.01653115 

0.2588230 

0.5000045 

0.70711  60 

0.8660375 

0.96593:9° 

I.  OCX 

58  0.01687 

15,15 

0.2630330,15 

0.50377145,15 

0.71019,60,15 

0.86820:75,15 

0.96705       .     . 

, 

59  0.01715 

15,30 

0.2672430,30 

o.50754!45-30 

0.71325160,30 

o  87036175,30 

0.96815       .     . 

. 

60  o.oi745  15,45 

0,2714430,45 

0.5112945,45 

0.71630:60,45 

0.8725075,45 

0.96923       .     . 

• 

